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Aims/hypothesis: Type 1 diabetes is characterised by a progressive decline in beta cell mass. This is also observed following implantation of pancreatic islet allografts, but there is no reliable information regarding the time course of beta cell loss. This is due to the limited availability of non-invasive pancreatic islet imaging techniques. We have previously described that dipeptidyl peptidase 6 (DPP6) is an alpha and beta cell-specific biomarker, and developed a camelid antibody (nanobody ‘4hD29’) against it. We demonstrated the possibility to detect DPP6-expressing cells by single-photon emission computed tomography (SPECT)/ computed tomography (CT), but the correlation between the number of cells grafted and the SPECT signal was not assessed. Here, we investigate whether the 4hD29 nanobody allows us to detect different amounts of human pancreatic islets implanted into immune-deficient mice. In addition, we also describe the adaptation of the probe for use with positron emission tomography (PET). Methods: DPP6 expression was assessed in human samples using tissue arrays and immunohistochemistry. The effect of the 4hD29 nanobody on cell death and glucose-stimulated insulin secretion was measured in EndoC-βH1 cells and in human islets using Hoechst/propidium iodide staining and an anti-insulin ELISA, respectively. We performed in vivo SPECT imaging on severe combined immunodeficient (SCID) mice transplanted with different amounts of EndoC-βH1 cells (2 × 106, 5 × 106 and 10 × 106 cells), human islets (1000 and 3000) or pancreatic exocrine tissue using 99mTc-labelled 4hD29 nanobody. This DPP6 nanobody was also conjugated to N-chlorosuccinimide (NCS)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), radiolabelled with either 67Ga (SPECT) or 68Ga (PET) and used in a proof-of-principle experiment to detect DPP6-expressing cells (Kelly neuroblastoma) grafted in SCID mice. Results: The DPP6 protein is mainly expressed in pancreatic islets. Importantly, the anti-DPP6 nanobody 4hD29 allows non-invasive detection of high amounts of EndoC-βH1 cells or human islets grafted in immunodeficient mice. This suggests that the probe must be further improved to detect lower numbers of islet cells. The 4hD29 nanobody neither affected beta cell viability nor altered insulin secretion in EndoC-βH1 cells and human islets. The conversion of 4hD29 nanobody into a PET probe was successful and did not alter its specificity. Conclusions/interpretation: These findings suggest that the anti-DPP6 4hD29 nanobody may become a useful tool for the quantification of human islet grafts in mice and, pending future development, islet mass in individuals with diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

Aims/hypothesis Transcription factor 7-like 2 (TCF7L2) is a Wnt-signalling-associated transcription factor. Genetic studies have clearly demonstrated that DNA polymorphisms within TCF7L2 confer the strongest known association with increased risk of type 2 diabetes. However, the impact of the TCF7L2 type-2-diabetes-associated rs7903146 T allele on biological function and morphology of human pancreatic islets is unknown.

To the Editor: Single-centre or collaborative efforts have been made to provide human pancreatic islets for use in basic science research. These include the Integrated Islet Distribution Program in the USA [1], the Human Islet Distribution Program for Basic Research at the University of Alberta in Canada [2] and the Oxford Islet Transplant Program in the UK. The European Consortium for Islet Transplantation (ECIT) set up the human islet distribution programme for basic research (http://ecit.dri-sanraffaele.org/en/activeprogram/index.html) in 2006 to distribute high-quality human islets to the European diabetes research community. The programme currently involves the San Raffaele Scientific Institute (Milan, Italy), the Hopitaux Universitaires de Geneve, (Geneve, Switzerland), Uppsala University Hospital (Uppsala, Sweden) and the Centre Hospitalier Regional Universitaire de Lille (France). The programme is coordinated by the San Raffaele Diabetes Research Institute and receives financial support from the JDRF. A web-based platform was developed in 2009 to coordinate and track programme activity (http://ecit.dri-sanraffaele.org/en/register/ index.html). Islet isolation and shipment were performed according to centre protocols. Here we report on programme activity over the past 5 years (from 1 November 2009 to 31 October 2014). One-hundred and twenty research groups have completed the online registration process. Of these, 66 users havemade at least one application to receive islets. A total of 93 applications were submitted from 13 countries: Switzerland (n=26), Sweden (n=13), Israel (n=13), Germany (n=12), France (n=9), Italy (n=7), Spain (n=4), Denmark (n=3), UK (n=2) and one each from Austria, Norway, Ireland and Finland. The total number of islets requested was 106.187 million (Fig. 1a). An ideal islet purity of ≥70% was requested in 97% of the applications. In terms of applications from individual research groups, the median number of requested islets was 20×10 (IQR 12.5×10) in 20 (IQR 37.5) shipments. Over the years the requests for human pancreatic islets from the programme for use in basic science research have increased substantially, with the number of islets requested reaching 31.042 million in 2014. The designated use of the islets in basic science, as stated on the request form to the ECIT centres, are shown in Fig. 1c, categorised into different areas of research. Over the first 5 years of the programme, a total of 31.643 million human islets have been distributed throughout 1,130 shipments (Fig 1a,b). The production and delivery of islets by the ECIT centres have been stable over the years. However, because of the burgeoning demand for islets and the lack of funding to cover additional islet isolation procedures, the R. Nano :R. Melzi :A. Mercalli : L. Piemonti (*) Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy e-mail: piemonti.lorenzo@hsr.it

To the Editor: Pancreatic agenesis is an extremely rare cause of permanent neonatal diabetes mellitus (NDM) in humans. It can be associated with severe intrauterine growth retardation as well as a plethora of abnormalities or malformations in the heart, biliary tract, gut, thyroid or brain [1–3]. To date, mutations in three genes have been shown to cause pancreatic agenesis, namely, PDX1 [1], PTF1A [2], and most recently GATA6 [3], all three of which encode transcription factors. The GATA6 study reported heterozygous coding mutations in a dozen individuals with permanent NDM presumed to be due to pancreatic agenesis [3]. The genetic evidence supporting the pathogenicity of these mutations seemed quite strong as the reported mutations had arisen de novo and none of these mutations was present in the 1,000 Genome Project database [3]. The authors concluded that GATA6 may have a key role in human pancreatic development, with strong implications for beta cell regenerative medicine in diabetes [3]. In the

AIMS/HYPOTHESIS: Thiazolidinediones are reported to improve pancreatic islet morphology and beta-cell function in rodents, supporting the hypothesis of a direct action of thiazolidinediones on endocrine islet cells. In this study we examined the expression of the peroxisome proliferator-activated receptor gamma, a nuclear receptor that is activated by naturally occurring fatty acids and synthetic thiazolidinediones, in normal human endocrine pancreatic cells. METHODS: Human islets were isolated from pancreata harvested in ten brain-dead lean non-diabetic adult donors. We analysed the gene and protein expression of the human peroxisome proliferator-activated receptor gamma and evaluated the effects of peroxisome proliferator-activated receptor gamma agonist on insulin secretion in human islet preparations. RESULTS: The RT-PCR carried out on total RNA from four distinct human islet preparations demonstrated the presence of peroxisome proliferator-activated receptor gamma mRNA. Western blot analysis showed the consistent expression of peroxisome proliferator-activated receptor gamma protein. Peroxisome proliferator-activated receptor gamma was shown to be present in all three endocrine cell types studied (alpha, beta and delta cells) by immunohistochemistry. CONCLUSION/INTERPRETATION: We found that peroxisome proliferator-activated receptor gamma is highly expressed in human islet endocrine cells, both at the mRNA and protein levels. These results support the hypothesis of a direct influence of peroxisome proliferator-activated receptor gamma agonist on human pancreatic endocrine cells.

Diabetes is characterised by loss and dysfunction of the beta cell. A major goal of diabetes therapy is to promote the formation of new beta cells. Polymorphisms of T cell factor 7-like 2 (TCF7L2) are associated with type 2 diabetes, negatively regulating beta cell survival and function. Here, we provide evidence for a role of TCF7L2 in beta cell proliferation and regeneration. Pancreatic sections from three mouse models (high-fat diet, exendin-4 and streptozotocin-treated mice) and from healthy individuals and patients with type 2 diabetes were used to investigate the association of beta cell regeneration and TCF7L2 levels. To analyse a direct effect of TCF7L2 on duct cell to beta cell conversion, TCF7L2 was overexpressed in isolated exocrine cells. TCF7L2 levels correlated with beta cell compensation during high-fat diet feeding. TCF7L2 was increased together with pancreatic duct cell proliferation and differentiation. Small islet-like cell clusters (ICCs) that contained TCF7L2 originated in the vicinity of the ductal epithelium. In human isolated exocrine tissue, TCF7L2 overexpression induced proliferation of pancreatic duct cells and ICC formation next to duct cells, an effect dependent on the JAK2/STAT3 pathway. The present study demonstrates that TCF7L2 overexpression fosters beta cell regeneration. Our findings imply correlation of TCF7L2 levels and new beta cell formation.

Unpredictability of islet isolation outcome remains a frustrating and costly issue in the clinical implementation of islet transplantation. The aim of this experimental study was to test the hypothesis that the donor's insulin secretory reserve, an in vivo surrogate of functional pancreatic mass, is correlated with the outcome of islet isolation.Insulin secretory reserve was evaluated in 28 healthy adult minipigs prior to pancreatectomy and islet isolation. Blood glucose and insulinaemia were measured before and 1, 3, 5, 10, 15, 30, 60 and 90 min after glucose infusion. Following total pancreatectomy, islet isolation was performed according to Ricordi's semi-automated method, and the total number of islets obtained was determined. Fasting blood glucose, insulinaemia, acute insulin response (AIR), maximal insulinaemia and the glucose decay constant (K(G)) were calculated, and possible associations with the outcome of islet isolation were assessed.AIR and maximal insulinaemia after glucose injection were correlated with the outcome of islet isolation (p0.01). Mean values for AIR and maximal insulinaemia were significantly different between animals in which islet isolation was successful (n=11) vs those in which it was unsuccessful (n=17) (77.6+/-13.7 microU/ml vs 42.3+/-7.8 microU/ml, p0.05; 144.7+/-21.6 microU/ml vs 71.9+/-10.4 microU/ml, p0.05, respectively).This study suggests that the donor's pancreatic endocrine mass, as estimated by AIR, is a major determinant of the outcome of islet isolation in large mammals. Our results may explain the frustrating variability of human islet isolation outcome and could lead to a new approach for optimising the selection of brain-dead and/or living pancreas donors.

Whether excess glucose (glucotoxicity) and excess non-esterified fatty acids (lipotoxicity) act synergistically or separately to alter beta-cell function on Type 2 diabetes remains controversial. We examined the influence of non-esterified fatty acids, with or without concomitant increased glucose concentrations, on human islet function and on the expression of genes involved in lipid metabolism.Human islets isolated from non-diabetic and non-obese donors were cultured with 5.5, 16 or 30 mmol/l glucose, and when appropriate with 1 or 2 mmol/l non-esterified fatty acids. After 48 h, glucose-stimulated insulin secretion, insulin content, triglyceride content and expression of different genes were evaluated.Non-esterified fatty acids decreased glucose-stimulated insulin secretion, insulin content and increased triglyceride content of human isolated islets, independently from the deleterious effect of glucose. Increased glucose concentrations also decreased glucose-stimulated insulin secretion and insulin content, but had no influence on triglyceride content. Glucose-stimulated insulin secretion of islets appeared to be significantly correlated with their triglyceride content. Glucose and non-esterified fatty acids modified the gene expression of carnitine palmitoyltransferase-I, acetyl-CoA carboxylase, acyl-CoA oxidase and uncoupling protein 2.In our model of isolated human islets, increased glucose and non-esterified fatty acids separately reproduced the two major beta-cell alterations observed in vivo, i.e. loss of glucose-stimulated insulin secretion and reduction in islet insulin content. Our results also suggest that this deleterious effect was, at least in part, mediated by modifications in lipid metabolism gene expression.

Aims/hypothesis. Vulnerability of pancreatic islets to oxygen free radicals and nitric oxide contributes to islet transplantation obstacles. This susceptibility can be linked to the low expression levels of antioxidant enzymes in islets. Our aim was to investigate the effect of overexpressing Cu/Zn superoxide dismutase in human islets through a simple procedure on the cytotoxic effects of two nitric oxide donors: 3-morpholinosydnonimine (SIN-1) and S-Nitroso-N-acetyl-d,l-penicillamine (SNAP). Methods. Cultured human islets and INS-1 rat-derived insulin-secreting cells were transfected by an E1-deleted adenovirus carrying Cu/Zn SOD cDNA under the control of a cytomegalovirus (CMV) promoter (AdSOD). The viability of the cells was tested by the WST-1 assay (Roche, Indianapolis, Ind., USA). Results. The AdSOD procedure allowed SOD activity to increase by twofold to threefold for 2 to 8 days following transfection. Adenovirus-driven SOD overexpression was associated with a significant reduction of SIN-1-induced cytotoxicity on human islets (69.9 ± 10.5 % protection at 200 μmol/l and 40.5 ± 8.9 % protection at 400 μmol/l) and INS-1 cells (82.2 ± 8.8 % protection at 200 μmol/l and 31.1 ± 5.8 % protection at 400 μmol/l). Protection against increasing doses of SNAP was AdSOD dose-dependent. Transfected islets released significantly more insulin than control islets in glucose-theophyllin-stimulated conditions, without or following exposure to SNAP. Conclusions/interpretation. We thus established that adenoviral-induced overexpression of Cu/Zn SOD can be beneficial to human islet endocrine function and resistance to nitric oxide cytotoxicity. These data could be relevant for the development of new strategies aimed at preventing NO-induced beta-cell damage in an islet transplantation setting. [Diabetologia (2000) 43: 625–631]

Susceptibility of pancreatic islets to oxidant stress may affect islet viability and contribute to primary non function of allo- or xenogenic grafts. We investigated the influence of overexpression of catalase (CAT) on the viability of human, porcine and rat islets, as well as INS-1 beta-cell line. Islets were transfected with a replication-deficient adenovirus vector containing human CAT cDNA under the control of the adenovirus major late promoter (AdCAT) or a vector containing no foreign gene (AdNull) and used as a control. Oxidant stress was induced 48 h later by xanthine oxidase-hypoxanthine (XO 25 mU/ml, HX 0.5 mmol/l) or hydrogen peroxide (100 or 250 micromol/l). Islet cell viability was assessed 72 h after CAT transfer by 4-[3-(4-Idophenyl)-2-(4 nitrophenyl)-2H-5-tetrazolio]-1,2,benzene disulphonate (WST-1) test. Baseline catalase activity was three to fourfold lower in porcine than in human islets. CAT activity was reproducibly increased 2.5- to 7-fold in AdCAT infected islets, at least for 13 days. Overall, AdCAT conferred on human and pig islets a protection of 26.1 +/- 6.1 and 21.2 +/- 9.8% on XOHX injury and 35.4 +/- 4.2 and 57.9 +/- 10.5% on H2O2 stress. Similarly, rat islet cells and INS-1 cells were protected on XOHX stress by 17.8 +/- 2.3 and 30.8 +/- 8.7%, respectively. AdNull had no effect. Basal and stimulated insulin secretion was preserved in AdCAT-transfected human islets despite a XOHX challenge. This study validates adenovirus-mediated catalase gene transfer as a realistic approach to reduce non specific inflammation effects on human or porcine islet grafts. Moreover the relevance of defense mechanisms, previously suggested in human islets, is here illustrated in porcine islets.