Laurianne Giovannoni

Unique arrangement of alpha- and beta-cells in human islets of Langerhans

OBJECTIVE It is generally admitted that the endocrine cell organization in human islets is different from that of rodent islets. However, a clear description of human islet architecture has not yet been reported. The aim of this work was to describe our observations on the arrangement of human islet cells. RESEARCH DESIGN AND METHODS Human pancreas specimens and isolated islets were processed for histology. Sections were analyzed by fluorescence microscopy after immunostaining for islet hormones and endothelial cells. RESULTS In small human islets (40–60 μm in diameter), β-cells had a core position, α-cells had a mantle position, and vessels laid at their periphery. In bigger islets, α-cells had a similar mantle position but were found also along vessels that penetrate and branch inside the islets. As a consequence of this organization, the ratio of β-cells to α-cells was constantly higher in the core than in the mantle part of the islets, and decreased with increasing islet diameter. This core-mantle segregation of islet cells was also observed in type 2 diabetic donors but not in cultured isolated islets. Three-dimensional analysis revealed that islet cells were in fact organized into trilaminar epithelial plates, folded with different degrees of complexity and bordered by vessels on both sides. In epithelial plates, most β-cells were located in a central position but frequently showed cytoplasmic extensions between outlying non–β-cells. CONCLUSIONS Human islets have a unique architecture allowing all endocrine cells to be adjacent to blood vessels and favoring heterologous contacts between β- and α-cells, while permitting homologous contacts between β-cells.

Resveratrol Potentiates Glucose-stimulated Insulin Secretion in INS-1E β-Cells and Human Islets through a SIRT1-dependent Mechanism

Resveratrol, a polyphenol compound, is known for its effects on energy homeostasis. With properties of energy sensors mediating effects of calorie restriction, sirtuins are targets of resveratrol. The mammalian sirtuin homolog SIRT1 is a protein deacetylase playing a role in glucose metabolism and islet function. Here, we investigated the effects of resveratrol and possible link with SIRT1 in β-cells. Insulinoma INS-1E cells and human islets were cultured with resveratrol before analyzing their physiological responses. Treatment of INS-1E cells for 24 h with 25 μm resveratrol resulted in marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated glycolytic flux, resulting in increased glucose oxidation, ATP generation, and mitochondrial oxygen consumption. Such changes correlated with up-regulation of key genes for β-cell function, i.e. Glut2, glucokinase, Pdx-1, Hnf-1α, and Tfam. In human islets, chronic resveratrol treatment similarly increased both the glucose secretory response and expression of the same set of genes, eventually restoring the glucose response in islets obtained from one type 2 diabetic donor. Overexpression of Sirt1 in INS-1E cells potentiated resveratrol effects on insulin secretion. Conversely, inhibition of SIRT1 achieved either by expression of an inactive mutant or by using the EX-527 inhibitor, both abolished resveratrol effects on glucose responses. Treatment of INS-1E cells with EX-527 also prevented resveratrol-induced up-regulation of Glut2, glucokinase, Pdx-1, and Tfam. Resveratrol markedly enhanced the glucose response of INS-1E cells and human islets, even after removal of the compound from the medium. These effects were mediated by and fully dependent on active SIRT1, defining a new role for SIRT1 in the regulation of insulin secretion.

Influence of donor age on islet isolation and transplantation outcome

BACKGROUND It has been suggested that the age of human organ donors might influence islet isolation and transplantation outcome in a negative way due to a decrease of in vivo function in islets isolated from older donors. METHODS We retrospectively analyzed 332 islet isolations according to donor age. We determined isolation outcome by islet yields, transplantation rates, and [beta]-cell function in vitro. Transplanted patients were divided into two groups depending on donor age (n=25 and n=31 patients for <=45- and >45-year-old donors, respectively). We assessed islet graft function by C-peptide/glucose ratio, [beta] score, secretory units of islets in transplantation index, and insulin independence rate at 1, 6, and 12 months after transplantation. RESULTS There was no difference in islet yields between the two groups (251,900+/-14,100 and 244,600+/-8400 islet equivalent for <=45- and >45-year-old donors, respectively). Transplantation rates and stimulation indices were similar in both groups as well. All islet graft function parameters were significantly higher at 1-month follow-up in patients who had received islets from younger donors. At 6-month follow-up after second or third injection and at 12-month follow-up, secretory units of islets in transplantation indices and C-peptide/glucose ratios were significantly higher in patients with donors aged 45 years or younger. CONCLUSIONS These data suggest that, despite similar outcomes of the isolation procedure, islet graft function is significantly influenced by donor age. These results may have important consequences in the definition of pancreas allocation criteria.

Immunosuppressive effects of streptozotocin-induced diabetes result in absolute lymphopenia and a relative increase of T regulatory cells

OBJECTIVE Streptozotocin (STZ) is the most widely used diabetogenic agent in animal models of islet transplantation. However, the immunomodifying effects of STZ and the ensuing hyperglycemia on lymphocyte subsets, particularly on T regulatory cells (Tregs), remain poorly understood. RESEARCH DESIGN AND METHODS This study evaluated how STZ-induced diabetes affects adaptive immunity and the consequences thereof on allograft rejection in murine models of islet and skin transplantation. The respective toxicity of STZ and hyperglycemia on lymphocyte subsets was tested in vitro. The effect of hyperglycemia was assessed independently of STZ in vivo by the removal of transplanted syngeneic islets, using an insulin pump, and with rat insulin promoter diphtheria toxin receptor transgenic mice. RESULTS Early lymphopenia in both blood and spleen was demonstrated after STZ administration. Direct toxicity of STZ on lymphocytes, particularly on CD8+ cells and B cells, was shown in vitro. Hyperglycemia also correlated with blood and spleen lymphopenia in vivo but was not lymphotoxic in vitro. Independently of hyperglycemia, STZ led to a relative increase of Tregs in vivo, with the latter retaining their suppressive capacity in vitro. The higher frequency of Tregs was associated with Treg proliferation in the blood, but not in the spleen, and higher blood levels of transforming growth factor-β. Finally, STZ administration delayed islet and skin allograft rejection compared with naive mice. CONCLUSIONS These data highlight the direct and indirect immunosuppressive effects of STZ and acute hyperglycemia, respectively. Thus, these results have important implications for the future development of tolerance-based protocols and their translation from the laboratory to the clinic.

Assessment of human islet labeling with clinical grade iron nanoparticles prior to transplantation for graft monitoring by MRI

Ex vivo labeling of islets with superparamagnetic iron oxide (SPIO) nanoparticles allows posttransplant MRI imaging of the graft. In the present study, we compare two clinical grade SPIOs (ferucarbotran and ferumoxide) in terms of toxicity, islet cellular uptake, and MRI imaging. Human islets (80–90% purity) were incubated for 24 h with various concentrations of SPIOs (14–280 μg/ml of iron). Static incubations were performed, comparing insulin response to basal (2.8 mM) or high glucose stimulation (16.7 mM), with or without cAMP stimulation. Insulin and Perls (assessment of iron content) staining were performed. Electronic microscopy analysis was performed. Labeled islets were used for in vitro or in vivo imaging in MRI 1.5T. Liver section after organ removal was performed in the same plane as MRI imaging to get a correlation between histology and radiology. Postlabeling islet viability (80 ± 10%) and function (in vitro static incubation and in vivo engraftment of human islets in nude mice) were similar in both groups. Iron uptake assessed by electron microscopy showed iron inclusions within the islets with ferucarbotran, but not with ferumoxide. MRI imaging (1.5T) of phantoms and of human islets transplanted in rats, demonstrated a strong signal with ferucarbotran, but only a weak signal with ferumoxide. Signal persisted for >8 weeks in the absence of rejection. An excellent correlation was observed between radiologic images and histology. The hepatic clearance of intraportally injected ferucarbotran was faster than that of ferumoxide, generating less background. A rapid signal decrease was observed in rejecting xenogeneic islets. According to the present data, ferucarbotran is the most appropriate of available clinical grade SPIOs for human islet imaging.

A functional circadian clock is required for proper insulin secretion by human pancreatic islet cells

To determine the impact of a functional human islet clock on insulin secretion and gene transcription.

Pancreatic α- and β-cellular clocks have distinct molecular properties and impact on islet hormone secretion and gene expression

A critical role of circadian oscillators in orchestrating insulin secretion and islet gene transcription has been demonstrated recently. However, these studies focused on whole islets and did not explore the interplay between α-cell and β-cell clocks. We performed a parallel analysis of the molecular properties of α-cell and β-cell oscillators using a mouse model expressing three reporter genes: one labeling α cells, one specific for β cells, and a third monitoring circadian gene expression. Thus, phase entrainment properties, gene expression, and functional outputs of the α-cell and β-cell clockworks could be assessed in vivo and in vitro at the population and single-cell level. These experiments showed that α-cellular and β-cellular clocks are oscillating with distinct phases in vivo and in vitro. Diurnal transcriptome analysis in separated α and β cells revealed that a high number of genes with key roles in islet physiology, including regulators of glucose sensing and hormone secretion, are differentially expressed in these cell types. Moreover, temporal insulin and glucagon secretion exhibited distinct oscillatory profiles both in vivo and in vitro. Altogether, our data indicate that differential entrainment characteristics of circadian α-cell and β-cell clocks are an important feature in the temporal coordination of endocrine function and gene expression.

Impact of the number of infusions on 2-year results of islet-after-kidney transplantation in the GRAGIL network.

Background. Insulin independence after islet transplantation is generally achieved after multiple infusions. However, single infusion would increase the number of recipients. Our aim was to evaluate the results of islet-after-kidney transplantation according to the number of infusions. Methods. Islets were isolated at the Geneva University, shipped, and transplanted into French patients from the Swiss-French GRAGIL network, on the “Edmonton” immunosuppression protocol between 2004 and 2010. Results. Nineteen patients were transplanted with 33 preparations. Fifteen patients reached 24 months follow-up; eight subjects were single-graft recipients and seven were double-graft recipients. Finally, single-graft recipients received a median of 5312 islet equivalents/kg (5186–6388) vs. 10,564 (10,054–11,375) for double-graft recipients (P=0.0003) with similar islet mass at first infusion. Insulin independence was achieved in five of eight single-graft subjects (62.5%) versus five of seven in double-graft subjects (71.4%), not significant. Median insulin independence duration was 4.7 (3.1–15.2) months after one infusion vs. 19 (9.6–20.8) months after two infusions (not significant). At 24 months posttransplant, comparing single- with double-graft patients, insulin doses were 0.23 (0.11–0.34) U/kg vs. 0.02 (0.0–0.23) U/kg, P=0.11; HbA1c was 6.5% (5.9%–6.8%) vs. 6.2% (5.9%–6.3%), P=0.16; and basal C-peptide was 302 (143–480) pmol/L vs. 599 (393–806) pmol/L, P=0.05. Only 37.5% of single-graft patients had a &bgr;-score ≥4 compared with 100% of double-graft patients (P=0.03). Two recipients experienced postinfusion bleeding, and two patients (13%) showed renal dysfunction in the absence of biopsy-proven rejection. Conclusions. One infusion achieves good glycemic control and sometimes insulin independence. However, double-graft patients remain insulin-free longer, tend to have lower HbA1c, and show better graft function 24 months after transplant.

Survival of Free and Encapsulated Human and Rat Islet Xenografts Transplanted into the Mouse Bone Marrow

Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.

Rapamycin impairs proliferation of transplanted islet β cells.

Background. The cause for a progressive attrition of islet graft function observed over the years after islet transplantation is not well defined but may be in part the result of adverse effects of immunosuppressive agents. In this study, we examined the effect of rapamycin, a key component of the immunosuppressive regimen, on &bgr;-cell replication of transplanted islets. Methods. Mice transplanted with rat islets under kidney capsule received bromodeoxyuridine for 7 days. Mice were treated with rapamycin or appropriate vehicle. &bgr;-cell replication was determined by double immunofluorescence staining for insulin and bromodeoxyuridine. For in vitro studies, apoptosis, glucose-stimulated insulin secretion, and proliferation were determined in islet cells incubated with EdU in the presence or absence of rapamycin. Results. In our islet transplant model, rapamycin impaired glucose tolerance and &bgr;-cell proliferation of transplanted and host islets. In vitro, rapamycin reduced glucose-stimulated insulin secretion and reversibly decreased &bgr;-cell replication. The inhibitory effect of rapamycin on &bgr;-cell proliferation was not due to the decrease in insulin release. Additionally, in islet cells, expression of cell cycle proteins was significantly modified by rapamycin, suggesting a blockade of cell cycle progression. Inhibition of p38MAPK partially reverted rapamycin effect on &bgr;-cell proliferation. Conclusion. Rapamycin, at concentration usually used to prevent islet graft rejection, is able to reduce the rate of &bgr;-cell proliferation in transplanted rat islets but also in host murine islets. These data suggest that the progressive islet graft dysfunction observed under immunosuppressive therapy may result in part from an impairment of &bgr;-cell regeneration.