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Objective: Although primarily secreted by the liver, Fibroblast Growth Factor 21 (FGF21) is also expressed in the pancreas, where its function remains unclear. This study aims to elucidate the role of the glucagon-FGF21 interaction in the metabolic benefits of SGLT2 inhibition (SGLT2i) and hypothesizes it is key to enhancing glucose and lipid metabolism in individuals with glucose intolerance or type 2 diabetes (T2D)., Methods: FGF21, FGF1R, and β-klotho expression in human pancreas was analysed by RNAscope, qPCR and immunofluorescent techniques. Glucose-stimulated insulin secretion (GSIS) assay was used to investigate the effects of recombinant FGF21 (rFGF21) on islets from donors with glucose intolerance or T2D. To explore the role of the glucagon-FGF21 axis in the benefits of SGLT2i, we used WT and Sglt2 knockout (KO) mice fed a chow diet (CD) or a high-fat diet (HFD) and chronically treated with vehicle or dapagliflozin., Results: Chronic rFGF21 treatment enhanced GSIS in islets from donors with glucose intolerance, with increased FGFR1 expression, suggesting FGF21's greater efficacy in the early stages of disease. In diet-induced insulin-resistant mice, dapagliflozin reduced postprandial glycaemia and elevated plasma glucagon and FGF21 levels. Sglt2 KO mice on a CD showed increased fasting plasma glucagon without changes in FGF21. In diet-induced insulin-resistant Sglt2 KO mice, elevated glucagon and FGF21 levels paralleled chronic dapagliflozin treatment, indicating similar metabolic adaptations in both models., Conclusion: Our findings indicate FGF21 as a crucial mediator in liver-pancreas crosstalk, improving lipid and glucose metabolism, enhancing pancreatic function, and potentiating the therapeutic efficacy of SGLT2i, thereby representing a target for prediabetes treatment., (© 2024 The Author(s). Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Aims/hypothesis: Insulitis, a hallmark of inflammation preceding autoimmune type 1 diabetes, leads to the eventual loss of functional beta cells. However, functional beta cells can persist even in the face of continuous insulitis. Despite advances in immunosuppressive treatments, maintaining functional beta cells to prevent insulitis progression and hyperglycaemia remains a challenge. The cannabinoid type 1 receptor (CB1R), present in immune cells and beta cells, regulates inflammation and beta cell function. Here, we pioneer an ex vivo model mirroring human insulitis to investigate the role of CB1R in this process., Methods: CD4 + T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) from male and female individuals at the onset of type 1 diabetes and from non-diabetic individuals, RNA was extracted and mRNA expression was analysed by real-time PCR. Single beta cell expression from donors with type 1 diabetes was obtained from data mining. Patient-derived human islets from male and female cadaveric donors were 3D-cultured in solubilised extracellular matrix gel in co-culture with the same donor PBMCs, and incubated with cytokines (IL-1β, TNF-α, IFN-γ) for 24-48 h in the presence of vehicle or increasing concentrations of the CB1R blocker JD-5037. Expression of CNR1 (encoding for CB1R) was ablated using CRISPR/Cas9 technology. Viability, intracellular stress and signalling were assayed by live-cell probing and real-time PCR. The islet function measured as glucose-stimulated insulin secretion was determined in a perifusion system. Infiltration of immune cells into the islets was monitored by microscopy. Non-obese diabetic mice aged 7 weeks were treated for 1 week with JD-5037, then euthanised. Profiling of immune cells infiltrated in the islets was performed by flow cytometry., Results: CNR1 expression was upregulated in circulating CD4 + T cells from individuals at type 1 diabetes onset (6.9-fold higher vs healthy individuals) and in sorted islet beta cells from donors with type 1 diabetes (3.6-fold higher vs healthy counterparts). The peripherally restricted CB1R inverse agonist JD-5037 arrested the initiation of insulitis in humans and mice. Mechanistically, CB1R blockade prevented islet NO production and ameliorated the ATF6 arm of the unfolded protein response. Consequently, cyto/chemokine expression decreased in human islets, leading to sustained islet cell viability and function., Conclusions/interpretation: These results suggest that CB1R could be an interesting target for type 1 diabetes while highlighting the regulatory mechanisms of insulitis. Moreover, these findings may apply to type 2 diabetes where islet inflammation is also a pathophysiological factor., Data Availability: Transcriptomic analysis of sorted human beta cells are from Gene Expression Omnibus database, accession no. GSE121863, available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3448161 ., (© 2024. The Author(s).)

In islet transplantation (ITx), primary graft function (PGF) or beta cell function measured early after last infusion is closely associated with long term clinical outcomes. We investigated the association between PGF and 5 year insulin independence rate in ITx and pancreas transplantation (PTx) recipients. This retrospective multicenter study included type 1 diabetes patients who underwent ITx in Lille and PTx in Nantes from 2000 to 2022. PGF was assessed using the validated Beta2-score and compared to normoglycemic control subjects. Subsequently, the 5 year insulin independence rates, as predicted by a validated PGF-based model, were compared to the actual rates observed in ITx and PTx patients. The study enrolled 39 ITx (23 ITA, 16 IAK), 209 PTx recipients (23 PTA, 14 PAK, 172 SPK), and 56 normoglycemic controls. Mean[SD] PGF was lower after ITx (ITA 22.3[5.2], IAK 24.8[6.4], than after PTx (PTA 38.9[15.3], PAK 36.8[9.0], SPK 38.7[10.5]), and lower than mean beta-cell function measured in normoglycemic control: 36.6[4.3]. The insulin independence rates observed at 5 years after PTA and PAK aligned with PGF predictions, and was higher after SPK. Our results indicate a similar relation between PGF and 5 year insulin independence in ITx and solitary PTx, shedding new light on long-term transplantation outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chetboun, Masset, Maanaoui, Defrance, Gmyr, Raverdy, Hubert, Bonner, Supiot, Kerleau, Blancho, Branchereau, Karam, Chelghaf, Houzet, Giral, Garandeau, Dantal, Le Mapihan, Jannin, Hazzan, Caiazzo, Kerr-Conte, Vantyghem, Cantarovich and Pattou.)

Insulin secretion from pancreatic β cells is regulated by multiple stimuli, including nutrients, hormones, neuronal inputs, and local signalling. Amino acids modulate insulin secretion via amino acid transporters expressed on β cells. The granin protein VGF has dual roles in β cells: regulating secretory granule formation and functioning as a multiple peptide precursor. A VGF-derived peptide, neuroendocrine regulatory peptide-4 (NERP-4), increases Ca 2+ influx in the pancreata of transgenic mice expressing apoaequorin, a Ca 2+ -induced bioluminescent protein complex. NERP-4 enhances glucose-stimulated insulin secretion from isolated human and mouse islets and β-cell-derived MIN6-K8 cells. NERP-4 administration reverses the impairment of β-cell maintenance and function in db/db mice by enhancing mitochondrial function and reducing metabolic stress. NERP-4 acts on sodium-coupled neutral amino acid transporter 2 (SNAT2), thereby increasing glutamine, alanine, and proline uptake into β cells and stimulating insulin secretion. SNAT2 deletion and inhibition abolish the protective effects of NERP-4 on β-cell maintenance. These findings demonstrate a novel autocrine mechanism of β-cell maintenance and function that is mediated by the peptide-amino acid transporter axis., (© 2023. The Author(s).)

Introduction: Nowadays, there are no strong diabetic pig models, yet they are required for various types of diabetes research. Using cutting-edge techniques, we attempted to develop a type 2 diabetic minipig model in this study by combining a partial pancreatectomy (Px) with an energetic overload administered either orally or parenterally., Methods: Different groups of minipigs, including Göttingen-like (GL, n = 17) and Ossabaw (O, n = 4), were developed. Prior to and following each intervention, metabolic assessments were conducted. First, the metabolic responses of the Göttingen-like (n = 3) and Ossabaw (n = 4) strains to a 2-month High-Fat, High-Sucrose diet (HFHSD) were compared. Then, other groups of GL minipigs were established: with a single Px (n = 10), a Px combined with a 2-month HFHSD (n = 6), and long-term intraportal glucose and lipid infusions that were either preceded by a Px (n = 4) or not (n = 4)., Results: After the 2-month HFHSD, there was no discernible change between the GL and O minipigs. The pancreatectomized group in GL minipigs showed a significantly lower Acute Insulin Response (AIR) (18.3 ± 10.0 IU/mL after Px vs. 34.9 ± 13.7 IU/mL before, p < .0005). In both long-term intraportal infusion groups, an increase in the Insulinogenic (IGI) and Hepatic Insulin Resistance Indexes (HIRI) was found with a decrease in the AIR, especially in the pancreatectomized group (IGI: 4.2 ± 1.9 after vs. 1.5 ± 0.8 before, p < .05; HIRI (×10 -5 ): 12.6 ± 7.9 after vs. 3.8 ± 4.3 before, p < .05; AIR: 24.4 ± 13.7 µIU/mL after vs. 43.9 ± 14.5 µIU/mL before, p < .005). Regardless of the group, there was no fasting hyperglycemia., Conclusions: In this study, we used pancreatectomy followed by long-term intraportal glucose and lipid infusions to develop an original minipig model with metabolic syndrome and early signs of glucose intolerance. We reaffirm the pig's usefulness as a preclinical model for the metabolic syndrome but without the fasting hyperglycemia that characterizes diabetes mellitus., (© 2023 The Authors. Endocrinology, Diabetes & Metabolism published by John Wiley & Sons Ltd.)

Islet transplantation is a unique paradigm in organ transplantation, since multiple donors are required to achieve complete insulin-independence. Preformed or de novo Donor Specific Antibodies (DSA) may target one or several donor islets, which adds complexity to the analysis of their impact. Adult patients with type 1 diabetes transplanted with pancreatic islets between 2005 and 2018 were included in a single-center observational study. Thirty-two recipients with available sera tested by solid-phase assays for anti-HLA antibodies during their whole follow-up were analyzed. Twenty-five recipients were islet-transplantation-alone recipients, and 7 islet-after-kidney recipients. Seven recipients presented with DSA at any time during follow-up (two with preformed DSA only, one with preformed and de novo DSA, 4 with de novo DSA only). Only islet-transplantation-alone recipients presented with de novo DSA. Three clinical trajectories were identified according to: 1/the presence of preformed DSA, 2/early de novo DSA or 3/late de novo DSA. Only late de novo DSA were associated with unfavorable outcomes, depicted by a decrease of the β-score. Islet transplantation with preformed DSA, even with high MFI values, is associated with favorable outcomes in our experience. On the contrary, de novo DSA, and especially late de novo DSA, may be associated with allograft loss., (© 2022. The Author(s).)

Painless and controlled on-demand drug delivery is the ultimate goal for the management of various chronic diseases, including diabetes. To achieve this purpose, microneedle patches are gaining increased attention. While degradable microneedle (MN) arrays are widely employed, the use of non-dissolving MN patches remains a challenge to overcome. In this study, we demonstrate that crosslinking gelatin methacrylate with polyethylene glycol diacrylate (PEGDA) is potent for engineering non-dissolving MN arrays. Incorporation of MoS 2 nanosheets as a photothermal component into MN hydrogels results in MNs featuring on-demand release properties. An optimized MoS 2 -MN array patch formed using a hydrogel solution containing 500 μg mL -1 of MoS 2 and photochemically crosslinked for 5 min shows required mechanical behavior under a normal compressive load to penetrate the stratum corneum of mice or pig skin and allows the delivery of macromolecular therapeutics such as insulin upon swelling. Using ex vivo and in vivo models, we show that the MoS 2 -MN patches can be used for loading and releasing insulin for therapeutic purposes. Indeed, transdermal administration of insulin loaded into MoS 2 -MN patches reduces blood glucose levels in C57BL/6 mice and mini-pigs comparably to subcutaneously injected insulin. We believe that this on-demand delivery system might alter the current insulin therapies and might be a potential approach for delivery of other proteins.

Diabetes cell therapy by human islet transplantation can restore an endogenous insulin secretion and normal glycaemic control in type 1 diabetic patients for as long as 10 years post transplantation. Before transplantation, each clinical islet preparation undergoes extensive in-vitro and in-vivo quality controls. The in-vivo quality control assay consists of transplanting human islets under the kidney capsule of immunocompromised mice. Currently, it is considered the best predictive factor to qualify clinical transplant efficiency. This chimeric model offers a wide area of study since it combines the possibility of producing not only quantitative but also a maximum of qualitative data. Today's technological advances allow us to obtain more accurate and stronger data from the animals used in research while ensuring their comfort and well-being throughout the protocol, including cage enrichment and pain treatment during and after surgery. As demonstrated in this valuable model, we are able to generate more usable results (Refine), while reducing the number of animals used (Reduce), by focusing on the development of ex-vivo analysis techniques (Replace), which clearly highlights the Burch and Russell 3Rs concept.

Background and Aims: Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) is an endogenous inhibitor of the LDL receptor (LDLR). Mendelian randomization studies suggest that PCSK9 deficiency increases diabetes risk, but the underlying mechanisms remain unknown. The aim of our study was to investigate whether PCSK9 or its inhibition may modulate beta cell function., Methods: We assessed PCSK9 and insulin colocalization in human pancreatic sections by epifluorescent and confocal microscopy. We also investigated the expression and the function of PCSK9 in the human EndoC-βH1 beta cell line, by ELISA and flow cytometry, respectively. PCSK9 was inhibited with Alirocumab or siRNA. LDLR expression and LDL uptake were assessed by flow cytometry., Results: PCSK9 was expressed and secreted from beta cells isolated from human pancreas as well as from EndoC-βH1 cells. PCSK9 secretion was enhanced by statin treatment. Recombinant PCSK9 decreased LDLR abundance at the surface of these cells, an effect abrogated by Alirocumab. Alirocumab as well as PCSK9 silencing increased LDLR expression at the surface of EndoC-βH1 cells. Neither exogenous PCSK9, nor Alirocumab, nor PCSK9 silencing significantly altered glucose-stimulated insulin secretion (GSIS) from these cells. High-low density lipoproteins (LDL) concentrations decreased GSIS, but the addition of PCSK9 or its inhibition did not modulate this phenomenon., Conclusions: While PCSK9 regulates LDLR abundance in beta cells, inhibition of exogenous or endogenous PCSK9 does not appear to significantly impact insulin secretion. This is reassuring for the safety of PCSK9 inhibitors in terms of beta cell function., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The alimentary limb has been proposed to be a key driver of the weight-loss-independent metabolic improvements that occur upon bariatric surgery. However, the one anastomosis gastric bypass (OAGB) procedure, consisting of one long biliary limb and a short common limb, induces similar beneficial metabolic effects compared to Roux-en-Y Gastric Bypass (RYGB) in humans, despite the lack of an alimentary limb. The aim of this study was to assess the role of the length of biliary and common limbs in the weight loss and metabolic effects that occur upon OAGB. OAGB and sham surgery, with or without modifications of the length of either the biliary limb or the common limb, were performed in Gottingen minipigs. Weight loss, metabolic changes, and the effects on plasma and intestinal bile acids (BAs) were assessed 15 days after surgery. OAGB significantly decreased body weight, improved glucose homeostasis, increased postprandial GLP-1 and fasting plasma BAs, and qualitatively changed the intestinal BA species composition. Resection of the biliary limb prevented the body weight loss effects of OAGB and attenuated the postprandial GLP-1 increase. Improvements in glucose homeostasis along with changes in plasma and intestinal BAs occurred after OAGB regardless of the biliary limb length. Resection of only the common limb reproduced the glucose homeostasis effects and the changes in intestinal BAs. Our results suggest that the changes in glucose metabolism and BAs after OAGB are mainly mediated by the length of the common limb, whereas the length of the biliary limb contributes to body weight loss. NEW & NOTEWORTHY Common limb mediates postprandial glucose metabolism change after gastric bypass whereas biliary limb contributes to weight loss.

Unveiling the key pathways underlying postnatal beta-cell proliferation can be instrumental to decipher the mechanisms of beta-cell mass plasticity to increased physiological demand of insulin during weight gain and pregnancy. Using transcriptome and global Serine Threonine Kinase activity (STK) analyses of islets from newborn (10 days old) and adult rats, we found that highly proliferative neonatal rat islet cells display a substantially elevated activity of the mitogen activated protein 3 kinase 12, also called dual leucine zipper-bearing kinase (Dlk). As a key upstream component of the c-Jun amino terminal kinase (Jnk) pathway, Dlk overexpression was associated with increased Jnk3 activity and was mainly localized in the beta-cell cytoplasm. We provide the evidence that Dlk associates with and activates Jnk3, and that this cascade stimulates the expression of Ccnd1 and Ccnd2, two essential cyclins controlling postnatal beta-cell replication. Silencing of Dlk or of Jnk3 in neonatal islet cells dramatically hampered primary beta-cell replication and the expression of the two cyclins. Moreover, the expression of Dlk, Jnk3, Ccnd1 and Ccnd2 was induced in high replicative islet beta cells from ob/ob mice during weight gain, and from pregnant female rats. In human islets from non-diabetic obese individuals, DLK expression was also cytoplasmic and the rise of the mRNA level was associated with an increase of JNK3, CCND1 and CCND2 mRNA levels, when compared to islets from lean and obese patients with diabetes. In conclusion, we find that activation of Jnk3 signalling by Dlk could be a key mechanism for adapting islet beta-cell mass during postnatal development and weight gain.

Human Endogenous Retrovirus W Envelope (HERV-W ENV) mRNA or protein can be found in peripheral blood mononuclear cells (PBMCs) and exocrine pancreas of patients with type 1 diabetes (T1D). Further, previous observations have shown an association between enteroviral infection and development of T1D; specifically, coxsackievirus-B (CV-B) has been detected in the blood and pancreas of patients with T1D. Notably, viruses can activate HERV-W expression. Hence, we evaluated the effect of CV-B4 infection on HERV-W ENV mRNA expression. Primary human pancreatic ductal cells were obtained from five brain-dead donors. In the pancreatic cells of three donors, the HERV-W ENV mRNA level measured using RT-qPCR was upregulated upon CV-B4 infection. The HERV-W ENV protein was detected in the infected cells using the immunoblot assay. In human PBMCs inoculated with CV-B4 or when CV-B4 was incubated with an enhancing serum, the HERV-W ENV mRNA level was higher than the background RNA level. In monocyte-derived macrophages obtained from 5 of 13 donors, the HERV-W ENV mRNA level was higher in cultures inoculated with CV-B4 than in the control. Therefore, CV-B4 can upregulate or induce the transcription of a certain HERV-W ENV copy (or copies) in primary cell cultures, such as monocytes, macrophages, and pancreatic cells.

Studies implicating sodium-glucose cotransporter 2 (SGLT2) inhibitors in glucagon secretion by pancreatic α-cells reported controversial results. We hypothesized that interindividual heterogeneity in SGLT2 expression and regulation may affect glucagon secretion by human α-cells in response to SGLT2 inhibitors. An unbiased RNA-sequencing analysis of 207 donors revealed an unprecedented level of heterogeneity of SLC5A2 expression. To determine heterogeneity of SGLT2 expression at the protein level, the anti-SGLT2 antibody was first rigorously evaluated for specificity, followed by Western blot and immunofluorescence analysis on islets from 10 and 12 donors, respectively. The results revealed a high interdonor variability of SGLT2 protein expression. Quantitative analysis of 665 human islets showed a significant SGLT2 protein colocalization with glucagon but not with insulin or somatostatin. Moreover, glucagon secretion by islets from 31 donors at low glucose (1 mmol/L) was also heterogeneous and correlated with dapagliflozin-induced glucagon secretion at 6 mmol/L glucose. Intriguingly, islets from three donors did not secrete glucagon in response to either 1 mmol/L glucose or dapagliflozin, indicating a functional impairment of the islets of these donors to glucose sensing and SGLT2 inhibition. Collectively, these data suggest that heterogeneous expression of SGLT2 protein and variability in glucagon secretory responses contribute to interindividual differences in response to SGLT2 inhibitors., (© 2020 by the American Diabetes Association.)

Objective: The long-term outcome of allogenic islet transplantation is unknown. The aim of this study was to evaluate the 10-year outcome of islet transplantation in patients with type 1 diabetes and hypoglycemia unawareness and/or a functioning kidney graft., Research Design and Methods: We enrolled in this prospective parallel-arm cohort study 28 subjects with type 1 diabetes who received islet transplantation either alone (ITA) or after a kidney graft (IAK). Islet transplantation consisted of two or three intraportal infusions of allogenic islets administered within (median [interquartile range]) 68 days (43-92). Immunosuppression was induced with interleukin-2 receptor antibodies and maintained with sirolimus and tacrolimus. The primary outcome was insulin independence with A1C ≤6.5% (48 mmol/mol). Secondary outcomes were patient and graft survival, severe hypoglycemic events (SHEs), metabolic control, and renal function., Results: The primary outcome was met by (Kaplan-Meier estimates [95% CI]) 39% (22-57) and 28% (13-45) of patients 5 and 10 years after islet transplantation, respectively. Graft function persisted in 82% (62-92) and 78% (57-89) of case subjects after 5 and 10 years, respectively, and was associated with improved glucose control, reduced need for exogenous insulin, and a marked decrease of SHEs. ITA and IAK had similar outcomes. Primary graft function, evaluated 1 month after the last islet infusion, was significantly associated with the duration of graft function and insulin independence., Conclusions: Islet transplantation with the Edmonton protocol can provide 10-year markedly improved metabolic control without SHEs in three-quarters of patients with type 1 diabetes, kidney transplanted or not., (© 2019 by the American Diabetes Association.)

Background: Antithrombin (AT) III physiological levels are decreased during septic shock and supplementation therapy could therefore be beneficial., Objective: We hypothesized that the use of recombinant human AT could reduce disseminated intravascular coagulation (DIC) occurrence., Methods: We conducted a randomized open label controlled experimental study. Ten female "Large White" pigs were challenged with i.v. infusion of Escherichia coli endotoxin. Two groups of 5 pigs were randomly assigned to receive either recombinant human AT 100 U/kg over 30 min (ATryn group) or 0.9% saline (control group). AT III levels, coagulation, hemostasis, inflammation parameters, hemodynamics, and microcirculatory parameters were measured over a 5-h period. Immediately after euthanasia, kidneys were withdrawn for histology evaluation. Statistical analysis was performed with nonparametric tests and Dunn's test for multiple comparisons., Results: AT III activity was significantly higher in the ATryn group than in the control group from 60% (213% [203-223] vs. 104% [98-115], P = 0.008, respectively) to 300 min (115% [95-124] vs. 79% [67-93], P = 0.03). Recombinant human AT supplementation had no impact on hemodynamics, microcirculatory parameters, and sequential changes of coagulation parameters (platelet count, fibrinogen level, thrombin-AT complexes, and von Willebrand factor). Interleukin 6 and tumor necrosis factor α values were statistically the same for both groups throughout the study. Percentage of thrombosed glomeruli and percentage of thrombosed capillary in glomerulus were not significantly different between both groups., Conclusions: In our model of endotoxic shock, a single low dose of recombinant human AT did not prevent DIC occurrence, severity, inflammatory profile, or hemodynamic alterations.

The newest classes of anti-diabetic agents include sodium-glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide 1 receptor (GLP1R) agonists. The SGLT2 inhibitor dapagliflozin reduces glucotoxicity by glycosuria but elevates glucagon secretion. The GLP1R agonist liraglutide inhibits glucagon; therefore, we hypothesize that the cotreatment of dapagliflozin with liraglutide could reduce hyperglucagonemia and hyperglycemia. Here we use five complementary models: human islet cultures, healthy mice, db/db mice, diet-induced obese (DIO) mice, and somatostatin receptor-2 (SSTR2) KO mice. A single administration of liraglutide and dapagliflozin in combination improves glycemia and reduces dapagliflozin-induced glucagon secretion in diabetic mice. Chronic treatment with liraglutide and dapagliflozin produces a sustainable reduction of glycemia compared with each drug alone. Moreover, liraglutide reduces dapagliflozin-induced glucagon secretion by enhancing somatostatin release, as demonstrated by SSTR2 inhibition in human islets and in mice. Collectively, these data provide mechanistic insights into how intra-islet GLP1R activation is critical for the regulation of glucose homeostasis., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

In December 2018, Diabetes and Diabetologia began requiring authors of papers reporting data obtained from studies on human islets to report critical characteristics of the human islets used for research. The islet community was asked to provide feedback on it. Here is the contribution by the European Consortium for Islet Transplantation., (© 2019 by the American Diabetes Association.)

Coxsackievirus-B4 (CV-B4) E2 can persist in the pancreatic ductal-like cells (Panc-1 cell line), which results in an impaired differentiation of these cells into islet-like cell aggregates (ICA). In this study, primary pancreatic ductal cells obtained as a by-product of islet isolation from the pancreas of seven brain-dead adults were inoculated with CV-B4 E2, followed-up for 29 days, and the impact was investigated. Viral titers in culture supernatants were analyzed throughout the culture. Intracellular viral RNA was detected by RT-PCR. Levels of ductal cell marker CK19 mRNA and of insulin mRNA were evaluated by qRT-PCR. The concentration of c-peptide in supernatants was determined by ELISA. Ductal cells exposed to trypsin and serum-free medium formed ICA and resulted in an increased insulin secretion. Ductal cells from five brain-dead donors were severely damaged by CV-B4 E2, whereas the virus persisted in cultures of cells obtained from the other two. The ICAs whose formation was induced on day 14 post-inoculation were scarce and appeared tiny in infected cultures. Also, insulin mRNA expression and c-peptide levels were strongly reduced compared to the controls. In conclusion, CV-B4 E2 lysed human primary pancreatic ductal cells or persisted in these cells, which resulted in the impairment of differentiation into insulin-producing cells.

Background: Total pancreatectomy with intraportal islet autotransplantation (TPIAT) rather than partial pancreatectomy could represent a major shift in the management of patients with resectable pancreatic ductal adenocarcinoma (PDAC) when risks of postoperative pancreatic fistula are well identified. This approach provides a theoretical risk of tumor cell dissemination when islet cells are transplanted into the portal vein. Our objective was to explore the safety of TPIAT in PDAC in a mouse preclinical model of subcutaneous xenotransplantation of human cells isolated from pancreatic specimen during partial pancreatectomy performed for PDAC., Methods: Patients requiring pancreatectomy for PDAC were prospectively included. Immunocompromised mice were transplanted with pancreatic cells isolated from the nonmalignant part of the surgical specimen (experimental group). Results were compared with pancreatic tumor implants (control group). Pancreatic grafts were explanted at 6 weeks for histological analyses., Results: Nine patients were included, and 31 mice were transplanted. In the experimental group, explants were microscopically devoid of tumor cell, and no metastasis was observed. In the control group, all explants were composed of tumor., Conclusions: We report in a preclinical model the absence of local and distant spreading of malignant cells after pancreatic islets xenograft isolated from PDAC patients. These data supports the oncological safety of TPIAT as valuable alternative to partial pancreatectomy for PDAC patients with a high risk of postoperative pancreatic fistula.

In allogenic islet transplantation (IT), high purity of islet preparations and low contamination by nonislet cells are generally favored. The aim of the present study was to analyze the relation between the purity of transplanted preparations and graft function during 5 years post-IT. Twenty-four patients with type 1 diabetes, followed for 5 years after IT, were enrolled. Metabolic parameters and daily insulin requirements were compared between patients who received islet preparations with a mean purity <50% (LOW purity) or ≥50% (HIGH purity). We also analyzed blood levels of carbohydrate antigen 19-9 (CA 19-9)-a biomarker of pancreatic ductal cells-and glucagon, before and after IT. At 5 years, mean hemoglobin A1c (HbA1c levels) (P = .01) and daily insulin requirements (P = .03) were lower in the LOW purity group. Insulin independence was more frequent in the LOW purity group (P < .05). CA19-9 and glucagon levels increased post-IT (P < .0001) and were inversely correlated with the degree of purity. Overall, our results suggest that nonislet cells have a beneficial effect on long-term islet graft function, possibly through ductal-to-endocrine cell differentiation. ClinicalTrial.gov NCT00446264 and NCT01123187., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Sodium lactate seemed to improve fluid balance and avoid fluid overload. The objective of this study was to determine if these beneficial effects can be at least partly explained by an improvement in disseminated intravascular coagulation (DIC)-associated renal microvascular thrombosis., Methods: Ancillary work of an interventional randomized open label controlled experimental study. Fifteen female "Large White" pigs (2 months old) were challenged with intravenous infusion of E. coli endotoxin. Three groups of five animals were randomly assigned to receive different fluids: a treatment group received sodium lactate 11.2% (SL group); an isotonic control group received 0.9% NaCl (NC group); a hypertonic control group, with the same amount of osmoles and sodium than SL group, received sodium bicarbonate 8.4% (SB group). Glomerular filtration rate (GFR) markers, coagulation and inflammation parameters were measured over a 5-h period. Immediately after euthanasia, kidneys were withdrawn for histological study. Statistical analysis was performed with nonparametric tests and the Dunn correction for multiple comparisons. A p < 0.05 was considered significant., Results: The direct immunofluorescence study revealed that the percentage of capillary sections thrombosed in glomerulus were significantly lesser in SL group [5 (0-28) %] compared to NC [64 (43-79) %, p = 0.01] and SB [64 (43-79), p = 0.03] groups. Alterations in platelet count and fibrinogen level occurred earlier and were significantly more pronounced in both control groups compared to SL group (p < 0.05 at 210 and 300 min). The increase in thrombin-antithrombin complexes was significantly higher in NC [754 (367-945) μg/mL; p = 0.03] and SB [463 (249-592) μg/mL; p = 0.03] groups than in SL group [176 (37-265) μg/mL]. At the end of the experiment, creatinine clearance was significantly higher in SL group [55.46 (30.07-67.85) mL/min] compared to NC group [1.52 (0.17-27.67) mL/min, p = 0.03]., Conclusions: In this study, we report that sodium lactate improves DIC-associated renal microvascular thrombosis and preserves GFR. These findings could at least partly explain the better fluid balance observed with sodium lactate infusion.

Aims: To understand better the control of insulin secretion by human β cells and to identify similarities to and differences from rodent models., Methods: Dynamic insulin secretion was measured in perifused human islets treated with pharmacological agents of known modes of action., Results: Glucokinase activation (Ro28-1675) lowered the glucose threshold for stimulation of insulin secretion to 1 mmol/L (G1), augmented the response to G3-G5 but not to G8-G15, whereas tolbutamide remained active in G20, which indicates that not all K ATP channels were closed by high glucose concentrations. An almost 2-fold greater response to G15 than to supramaximal tolbutamide in G3 or to KCl+diazoxide in G15 vs G3 quantified the contribution of metabolic amplification to insulin secretion. Both disruption (latrunculin-B) and stabilization (jasplakinolide) of microfilaments augmented insulin secretion without affecting metabolic amplification. Tolbutamide-induced insulin secretion was consistently greater in G10 than G3, with a threshold at 1 and maximum at 10 µmol/L tolbutamide in G10, vs 10 and 25 µmol/L in G3. Sulphonylurea effects were thus clearly glucose-dependent. Insulin secretion was also increased by inhibiting K channels other than K ATP channels: Kv or BK channels (tetraethylammonium), TASK-1 channels (ML-365) and SK4 channels (TRAM-34). Opening K ATP channels with diazoxide inhibited glucose-induced insulin secretion with half maximum inhibitory concentrations of 9.6 and 24 µmol/L at G7 and G15. Blockade of L-type Ca channels (nimodipine) abolished insulin secretion, whereas a blocker of T-type Ca channels (NNC-55-0396) was ineffective at specific concentrations. Blockade of Na channels (tetrodotoxin) did not affect glucose-induced insulin secretion., Conclusions: In addition to sharing a K ATP channel-dependent triggering pathway and a metabolic amplifying pathway, human and rodent β cells were found to display more similarities than differences in the control of insulin secretion., (© 2017 John Wiley & Sons Ltd.)

Background: Sodium lactate has been shown to improve hemodynamics and avoid fluid overload. The objective of this study was to confirm a beneficial effect on fluid balance with sodium lactate infusion and to specify whether the advantage of lactate is related to a negative chloride balance, its particular metabolism, or simply its energy load., Methods: This was an interventional, randomized, open-label, controlled experimental study. Fifteen female "large white" pigs (2 months old) were challenged with intravenous infusion of Escherichia coli endotoxin. Three groups of five animals were randomly assigned to receive different fluids: a treatment group received sodium lactate 11.2% (SL group); an isotonic control group received 0.9% NaCl (NC group); and a hypertonic control group, with the same amount of osmoles and sodium as the SL group, received sodium bicarbonate 8.4% (SB group). In order to provide the same energy load in the three groups, control groups were perfused with an equivalent energy supply. Statistical analysis was performed with non-parametric tests and the Dunn correction for multiple comparisons at p < 0.05., Results: Fluid and chloride balance, hemodynamics, oxygenation markers, and microcirculatory parameters were measured over a 5-h period. Cumulative fluid balance was significantly lower in the SL group (550 (415-800) mL; median (interquartile range)) compared to the NC group (1100 (920-1640) mL, p = 0.01) and the SB group (935 (790-1220) mL, p = 0.03). Hemodynamics, cardiac efficiency, and microcirculation were significantly enhanced in the SL group, resulting in a significant improvement in oxygen delivery (SL group 417 (305-565) mL/min/m 2 at 300 min versus the NC (207 (119-272) mL/min/m 2 , p = 0.01) and the SB (278, (211-315) mL/min/m 2 , p = 0.03) groups). Oxygenation markers (arterial oxygen partial pressure (PaO 2 )/inspired oxygen fraction (FiO 2 ), mixed venous oxygen saturation (SvO 2 ), and venoarterial carbon dioxide tension difference (Pv-aCO 2 ) were enhanced with sodium lactate infusion. Chloride balance was equivalent in both hypertonic groups and significantly reduced compared to the NC group., Conclusion: Sodium lactate infusion improves fluid balance and hemodynamics. The advantage of lactate does not seem to be explained by its energy load or by the induced negative chloride balance with subsequent water movements.

Human islet transplantation is a viable treatment option for type 1 diabetes mellitus (T1DM). However, pancreatic islet inflammation after transplantation induced by innate immune responses is likely to hinder graft function. This is mediated by incompatibility between islets and the blood interface, known as instant blood-mediated inflammatory reaction (IBMIR). Herein we hypothesized that portal venous administration of islet cells with human recombinant antithrombin (ATryn®), a serine protease inhibitor (serpin), which plays a central role in the physiological regulation of coagulation and exerts indirect anti-inflammatory activities, may offset coagulation abnormalities such as disseminated intravascular coagulation (DIC) and IBMIR. The current prospective, randomized experiment was conducted using an established preclinical pig model. Three groups were constituted for digested pancreatic tissue transplantation (0.15 ml/kg): control, NaCl 0.9% (n = 7); gold standard, heparin (25 UI/kg) (n = 7); and human recombinant ATryn® (500 UI/kg) (n = 7). Blood samples were collected over time (T0 to 24 h), and biochemical, coagulation, and inflammatory parameters were evaluated. In both the control and heparin groups, one animal died after a portal thrombosis, while no deaths occurred in the ATryn®-treated group. As expected, islet transplantation was associated with an increase in plasma IL-6 or TNF-α levels in all three groups. However, DIC was only observed in the control group, an effect that was suppressed after ATryn® administration. ATryn® administration increased antithrombin activity by 800%, which remained at 200% for the remaining period of the study, without any hemorrhagic complications. These studies suggest that coadministration of ATryn® and pancreatic islets via intraportal transplantation may be a valuable therapeutic approach for DIC without risk for islets and subjects.

Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1α, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment., Competing Interests: The authors have declared that no competing interests exist.

Gastro-intestinal exclusion by Roux-en-Y gastric bypass (RYGB) improves glucose metabolism, independent of weight loss. Although changes in intestinal bile trafficking have been shown to play a role, the underlying mechanisms are unclear. We performed RYGB in minipigs and showed that the intestinal uptake of ingested glucose is blunted in the bile-deprived alimentary limb (AL). Glucose uptake in the AL was restored by the addition of bile, and this effect was abolished when active glucose intestinal transport was blocked with phlorizin. Sodium-glucose cotransporter 1 remained expressed in the AL, while intraluminal sodium content was markedly decreased. Adding sodium to the AL had the same effect as bile on glucose uptake. It also increased postprandial blood glucose response in conscious minipigs following RYGB. The decrease in intestinal uptake of glucose after RYGB was confirmed in humans. Our results demonstrate that bile diversion affects postprandial glucose metabolism by modulating sodium-glucose intestinal cotransport., (Copyright © 2016 Elsevier Inc. All rights reserved.)

An early activation of coagulation and fibrinolysis occurs during sepsis, leading to the syndrome of disseminated intravascular coagulation (DIC). Obesity has been demonstrated to be a hypercoagulable and hypofibrinolytic state, but its impact on DIC has never been studied. In this study, we aimed to determine if obesity impairs DIC in an acute endotoxic shock model using minipigs. This was a prospective, comparative, and experimental ancillary study approved by the Animal Ethics Committee. Pigs were chosen as a clinically relevant species, resembling humans in coagulation reactions. Four groups of five "Yucatan" minipigs were studied: lean and obese control groups, a lean lipopolysaccharide (LPS) group receiving Escherichia coli endotoxin (LPS), and an obese LPS group receiving the same endotoxin dose. We measured standard coagulation parameters (prothrombin time [PT], platelet count, and fibrinogen levels), thrombin-antithrombin complexes, tissue-type plasminogen activator, and plasminogen activator inhibitor-1. All measurements were performed at baseline and 30, 60, 90, 150, and 300 min. Results were given as median with interquartile ranges. At baseline, platelet count (477 [428 - 532] G/L vs. 381 [307 - 442] G/L; P = 0.005) and fibrinogen levels (4.6 [3.8 - 5.2] g/L vs. 2 [1.8 - 2.9] g/L; P < 0.001) were significantly higher, whereas PT (80% [76% - 92%] vs. 96% [89% - 100%]; P = 0.01) was significantly lower in obese pigs compared with lean pigs. In the LPS groups, administration of endotoxin resulted in a typical hypokinetic shock with DIC. The decrease in coagulation parameters (PT, platelet count, and fibrinogen levels) and the increase in thrombin-antithrombin complexes (581 [382 - 1,057] μg/mL vs. 247 [125 - 369] μg/mL at 150 min; P = 0.03) were significantly more important in the obese LPS group compared with those in the lean LPS group. Concerning the fibrinolytic reaction, we found a slightly more elevated increase of plasminogen activator inhibitor-1 in the obese LPS group at 300 min (481 [365 - 617] ng/mL vs. 355 [209 - 660] ng/mL; P = 0.66). In our model of endotoxic shock, obese pigs developed a more severe DIC with a more severe procoagulant response.

Type 2 diabetes (T2D) is characterized by chronic hyperglycemia resulting from a deficiency in insulin signaling, because of insulin resistance and/or defects in insulin secretion; it is also associated with increases in glucagon and endogenous glucose production (EGP). Gliflozins, including dapagliflozin, are a new class of approved oral antidiabetic agents that specifically inhibit sodium-glucose co-transporter 2 (SGLT2) function in the kidney, thus preventing renal glucose reabsorption and increasing glycosuria in diabetic individuals while reducing hyperglycemia. However, gliflozin treatment in subjects with T2D increases both plasma glucagon and EGP by unknown mechanisms. In spite of the rise in EGP, T2D patients treated with gliflozin have lower blood glucose levels than those receiving placebo, possibly because of increased glycosuria; however, the resulting increase in plasma glucagon levels represents a possible concerning side effect, especially in a patient population already affected by hyperglucagonemia. Here we demonstrate that SGLT2 is expressed in glucagon-secreting alpha cells of the pancreatic islets. We further found that expression of SLC5A2 (which encodes SGLT2) was lower and glucagon (GCG) gene expression was higher in islets from T2D individuals and in normal islets exposed to chronic hyperglycemia than in islets from non-diabetics. Moreover, hepatocyte nuclear factor 4-α (HNF4A) is specifically expressed in human alpha cells, in which it controls SLC5A2 expression, and its expression is downregulated by hyperglycemia. In addition, inhibition of either SLC5A2 via siRNA-induced gene silencing or SGLT2 via dapagliflozin treatment in human islets triggered glucagon secretion through KATP channel activation. Finally, we found that dapagliflozin treatment further promotes glucagon secretion and hepatic gluconeogenesis in healthy mice, thereby limiting the decrease of plasma glucose induced by fasting. Collectively, these results identify a heretofore unknown role of SGLT2 and designate dapagliflozin an alpha cell secretagogue.

Background: Pancreatic islet transplantation offers a promising biotherapy for the treatment of type 1 diabetes, but this procedure has met significant challenges over the years. One such challenge is to address why primary graft function still remains inconsistent after islet transplantation. Several variables have been shown to affect graft function, but the impact of procedure-related complications on primary and long-term graft functions has not yet been explored., Methods: Twenty-six patients with established type 1 diabetes were included in this study. Each patient had two to three intraportal islet infusions to obtain 10,000 islet equivalent (IEQ)/kg in body weight, equaling a total of 68 islet infusions. Islet transplantation consisted of three sequential fresh islet infusions within 3 months. Islet infusions were performed surgically or under ultrasound guidance, depending on patient morphology, availability of the radiology suite, and patient medical history. Prospective assessment of adverse events was recorded and graded using "Common Terminology Criteria for adverse events in Trials of Adult Pancreatic Islet Transplantation.", Results: There were no deaths or patients dropouts. Early complications occurred in nine of 68 procedures. β score 1 month after the last graft and optimal graft function (β score ≥7) rate were significantly lower in cases of procedure-related complications (P = 0.02, P = 0.03). Procedure-related complications negatively impacted graft function (P = 0.009) and was an independent predictive factor of long-term graft survival (P = 0.033) in multivariate analysis., Conclusion: Complications occurring during radiologic or surgical intraportal islet transplantation significantly impair primary graft function and graft survival regardless of their severity.

Reliable assessment of islet viability, mass, and purity must be met prior to transplanting an islet preparation into patients with type 1 diabetes. The standard method for quantifying human islet preparations is by direct microscopic analysis of dithizone-stained islet samples, but this technique may be susceptible to inter-/intraobserver variability, which may induce false positive/negative islet counts. Here we describe a simple, reliable, automated digital image analysis (ADIA) technique for accurately quantifying islets into total islet number, islet equivalent number (IEQ), and islet purity before islet transplantation. Islets were isolated and purified from n = 42 human pancreata according to the automated method of Ricordi et al. For each preparation, three islet samples were stained with dithizone and expressed as IEQ number. Islets were analyzed manually by microscopy or automatically quantified using Nikon's inverted Eclipse Ti microscope with built-in NIS-Elements Advanced Research (AR) software. The AIDA method significantly enhanced the number of islet preparations eligible for engraftment compared to the standard manual method (p < 0.001). Comparisons of individual methods showed good correlations between mean values of IEQ number (r(2) = 0.91) and total islet number (r(2) = 0.88) and thus increased to r(2) = 0.93 when islet surface area was estimated comparatively with IEQ number. The ADIA method showed very high intraobserver reproducibility compared to the standard manual method (p < 0.001). However, islet purity was routinely estimated as significantly higher with the manual method versus the ADIA method (p < 0.001). The ADIA method also detected small islets between 10 and 50 µm in size. Automated digital image analysis utilizing the Nikon Instruments software is an unbiased, simple, and reliable teaching tool to comprehensively assess the individual size of each islet cell preparation prior to transplantation. Implementation of this technology to improve engraftment may help to advance the therapeutic efficacy and accessibility of islet transplantation across centers.

Introduction: Based on the potential interest in sodium lactate as an energy substrate and resuscitative fluid, we investigated the effects of hypertonic sodium lactate in a porcine endotoxic shock., Methods: Fifteen anesthetized, mechanically ventilated pigs were challenged with intravenous infusion of E. coli endotoxin. Three groups of five animals were randomly assigned to receive 5 mL/kg/h of different fluids: a treatment group received hypertonic sodium lactate 11.2% (HSL group); an isotonic control group receiving 0.9% NaCl (NC group); a hypertonic control group with the same amount of osmoles and sodium than HSL group receiving hypertonic sodium bicarbonate 8.4% (HSB group). Hemodynamic and oxygenation variables, urine output and fluid balance were measured at baseline and at 30, 60, 120, 210 and 300 min. Skin microvascular blood flow at rest and during reactive hyperemia was obtained using a laser Doppler flowmetry technique. Results were given as median with interquartile ranges., Results: Endotoxin infusion resulted in hypodynamic shock. At 300 min, hemodynamics and oxygenation were significantly enhanced in HSL group: mean arterial pressure (103 [81-120] mmHg vs. 49 [41-62] in NC group vs. 71 [60-78] in HSB group), cardiac index (1.6 [1.2-1.8] L/min/m2 vs. 0.9 [0.5-1.1] in NC group vs. 1.3 [0.9-1.6] in HSB group) and partial pressure of oxygen (366 [308-392] mmHg vs. 166 [130-206] in NC group vs. 277 [189-303] in HSB group). At the same time, microvascular reactivity was significantly better in HSL group with a lower venoarterial CO2 tension difference (5.5 [4-10] mmHg vs. 17 [14-25] in NC group vs. 14 [12-15] in HSB group). The cumulative fluid balance was lower in HSL group (-325 [-655; -150] mL) compared to NC (+560 [+230; +900] mL, p = 0.008) and HSB (+185 [-110; +645] mL, p = 0.03) groups., Conclusions: In our hypodynamic model of endotoxic shock, infusion of hypertonic sodium lactate improves hemodynamic and microvascular reactivity with a negative fluid balance and a better oxygenation.

Studies exploring the influence of obesity on septic shock remain limited and controversial. Pigs were chosen as a clinically relevant species, resembling to humans in various functions. We hypothesize obesity may impair porcine acute endotoxic shock. Four groups of five "Yucatan" minipigs were studied: lean and obese control groups, lean lipopolysaccharide (LPS) group receiving Escherichia coli endotoxin (LPS) and obese LPS group receiving the same endotoxin dose. We measured hemodynamic and oxygenation parameters, skin microvascular blood flow at rest and during reactive hyperemia, von Willebrand factor, tumor necrosis factor α, and interleukin 6. All measurements were performed at baseline and at 30, 60, 90, 150, and 300 min. Results were given as median with 25th to 75th interquartile range. Control groups remained stable during the study period. In LPS groups, administration of endotoxin resulted in a typical hypokinetic shock. In obese LPS group at 300 min, we observed a significant impairment of cardiac index (1.2 [1.06-1.45] vs. 1.7 [1.57-1.97] L/min per m, P = 0.008) compared with the lean LPS group; moreover, pulmonary hypertension (mean arterial pressure: 42 [39-47] vs. 32 [28-34] mmHg, P = 0.008), hypoxemia (partial pressure of oxygen: 216 [178-262] vs. 325 [285-414] mmHg, P = 0.02), and lactate levels (5.8 [4.2-6.8] vs. 3.9 [2.2-5.5] mmol/L, P = 0.04) were significantly higher compared with the lean LPS group. Throughout the study, rest flow and peak flow during reactive hyperemia were more decreased in the obese LPS group. Compared with the lean LPS group, tumor necrosis factor α levels at 60 min (269 [178-428] vs. 126 [105-166] ng/mL, P = 0.03) and interleukin 6 levels at 300 min (101 [61-142] vs. 52 [36-64] ng/mL, P = 0.03) were significantly higher in the obese LPS group. In our model of endotoxic shock, obese pigs developed a more severe hemodynamic failure with pronounced microcirculatory dysfunction and proinflammatory response.

Background: Gastric bypass in obese patients induces a dramatic increase of postprandial insulin and glucagon-like peptide-1 (GLP-1) secretion, independently of weight loss. We explored postprandial insulin and GLP-1 secretion in nonobese minipigs before and after RYGB., Methods: Lean adult Göttingen minipigs (n = 7) were submitted to an open gastric bypass surgery mimicking the clinical procedure in humans (30-cm(3) gastric pouch/150-cm alimentary limb/70-cm biliary limb). All animals were evaluated at baseline and then 10 and 30 days after surgery. At each time point, serum glucose, insulin, GLP-1 and D-xylose levels were measured 3 h after a standardized mixed meal., Results: Weight remained stable during follow-up. Insulin and GLP-1 responses to the test meal were dramatically and similarly increased at 10 days and 1 month after RYGB. Maximal postprandial insulin and GLP-1 levels were 16.3 ± 1.7 mIU/l and 71.7 ± 16.5 pmol/l at baseline, 111.5 ± 38.9 mIU/l and 320.8 ± 84.0 pmol/l at 10 days and 96.6 ± 10.4 mIU/l and 297.3 ± 79.1 pmol/l at 1 month, respectively. D-Xylose absorption remained unchanged before and after surgery., Conclusions: RYGB induced a dramatic increase of postprandial insulin and GLP-1 secretion in nonobese minipigs. This preclinical model could help to understand the underlying metabolic effects of RYGB, focusing on the role of postsurgical anatomical rearrangement, especially duodenojejunal exclusion and ileal brake. This study supports the use of RYGB in diabetic nonobese patients in absence of obesity.

Elevation of the dietary saturated fatty acid palmitate contributes to the reduction of functional beta cell mass in the pathogenesis of type 2 diabetes. The diabetogenic effect of palmitate is achieved by increasing beta cell death through induction of the endoplasmic reticulum (ER) stress markers including activating transcription factor 3 (Atf3) and CAAT/enhancer-binding protein homologous protein-10 (Chop). In this study, we investigated whether treatment of beta cells with the MS-275, a HDAC1 and HDAC3 activity inhibitor which prevents beta cell death elicited by cytokines, is beneficial for combating beta cell dysfunction caused by palmitate. We show that culture of isolated human islets and MIN6 cells with MS-275 reduced apoptosis evoked by palmitate. The protective effect of MS-275 was associated with the attenuation of the expression of Atf3 and Chop. Silencing of HDAC3, but not of HDAC1, mimicked the effects of MS-275 on the expression of the two ER stress markers and apoptosis. These data point to HDAC3 as a potential drug target for preserving beta cells against lipotoxicity in diabetes.

Preservation of beta cell against apoptosis is one of the therapeutic benefits of the glucagon-like peptide-1 (GLP1) antidiabetic mimetics for preserving the functional beta cell mass exposed to diabetogenic condition including proinflammatory cytokines. The mitogen activated protein kinase 10 also called c-jun amino-terminal kinase 3 (JNK3) plays a protective role in insulin-secreting cells against death caused by cytokines. In this study, we investigated whether the JNK3 expression is associated with the protective effect elicited by the GLP1 mimetic exendin 4. We found an increase in the abundance of JNK3 in isolated human islets and INS-1E cells cultured with exendin 4. Induction of JNK3 by exendin 4 was associated with an increased survival of INS-1E cells. Silencing of JNK3 prevented the cytoprotective effect of exendin 4 against apoptosis elicited by culture condition and cytokines. These results emphasize the requirement of JNK3 in the antiapoptotic effects of exendin 4.

The role of enteroviruses, especially Coxsackievirus B (CVB), in type 1 diabetes is suspected, but the mechanisms of the virus-induced or aggravated pathogenesis of the disease are unknown. The hypothesis of an enterovirus-induced disturbance of pancreatic β-cells regeneration has been investigated in the human system. The infection of human pancreas ductal cells and pancreatic duct cell line, PANC-1, with CVB4E2 has been studied. Primary ductal cells and PANC-1 cells were infectable with CVB4E2 and a RT-PCR assay without extraction displayed that a larger proportion of cells harbored viral RNA than predicted by the detection of the viral capsid protein VP1 by indirect immunofluorescence. The detection of intracellular positive- and negative-strands of enterovirus genomes in cellular extracts by RT-PCR and the presence of infectious particles in supernatant fluids during the 37 weeks of monitoring demonstrated that CVB4E2 could persist in the pancreatic duct cell line. A persistent infection of these cells resulted in an impaired expression of Pdx1, a transcription factor required for the formation of endocrine pancreas, and a disturbed formation of islet-like cell aggregates of which the viability was decreased. These data support the hypothesis of an impact of enteroviruses onto pancreatic ductal cells which are involved in the renewal of pancreatic β-cells.

The liver may not be an optimal site for islet transplantation due to obstacles by an instant blood-mediated inflammatory response (IBMIR), and low revascularization of transplanted islets. Therefore, intramuscular islet transplantation (IMIT) offers an attractive alternative, based on its simplicity, enabling easier access for noninvasive graft imaging and cell explantation. In this study, we explored the outcome of autologous IMIT in the minipig (n = 30). Using the intramuscular injection technique, we demonstrated by direct histological evidence the rapid revascularization of islets autotransplanted into the gracilius muscle. Islet survival assessment was performed using immunohistochemistry staining for insulin and glucagon up to a period of 6 months. Furthermore, we showed the crucial role of minimizing mechanical trauma to the myofibers and limiting exocrine contamination. Intramuscular islet graft function after transplantation was confirmed by documenting the acute insulin response to intravenous glucose in 5/11 pancreatectomized animals. Graft function after IMIT remained however significantly lower than the function measured in 12 out of 18 minipigs who received a similar islet volume in the liver through intraportal infusion. Collectively, these results demonstrated in a clinically relevant preclinical model, suggest IMIT as a promising alternative to intraportal infusion for the transplantation of β cells in certain medical situations., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Aims/hypothesis: In this study, we used an immunodeficient mouse model to explore, in vivo, the longitudinal adaptation of human islets to an obesogenic environment., Methods: Non-diabetic Rag2 (-/-) mice (n = 61) were transplanted with human islets (400 islet equivalents [IEQ]) from six pancreases: four non-diabetic and two with overt metabolic dysfunction (older, high HbA(lc) or history of diabetes). Animals were fed for 12 weeks with a control or high-fat diet (HFD), and followed for weight, serum triacylglycerol, fasting blood glucose and human C-peptide. After the mice were killed, human grafts and the endogenous pancreas were analysed for endocrine volume, distribution of beta and alpha cells, and proliferation., Results: Transplanted mice on an HFD gained significantly more weight (p < 0.001) and had higher fasting glycaemia (2-12 weeks; p = 0.0002) and consistently higher fasting human C-peptide levels (2-12 weeks; p = 0.04) compared with those on the control diet. Histology demonstrated doubling of human islet graft volume at 12 weeks in animals on the HFD and increased beta cell volume (p < 0.001), but no change in alpha cell volume. Human islet function (hyperbolic product HOMA2%BS) at 12 weeks was four times lower in HFD animals (p < 0.001 vs controls) because of insufficient beta cell adaptation to decreased (70%) sensitivity (HOMA%S). Human islets obtained from donors with metabolic dysfunction failed to adapt to the HFD., Conclusions/interpretation: This longitudinal study provides direct evidence that human islets adapt both endocrine and beta cell mass, function and gene expression to obesity in vivo. The present model will facilitate the identification of mechanisms by which human islets adapt to obesity in vivo and the cell type(s) responsible, and factors predisposing human beta cells to decompensation.

Regulated associated protein of mTOR (Raptor) and rapamycin-insensitive companion of mTOR (rictor) are two proteins that delineate two different mTOR complexes, mTORC1 and mTORC2 respectively. Recent studies demonstrated the role of rictor in the development and function of β-cells. mTORC1 has long been known to impact β-cell function and development. However, most of the studies evaluating its role used either drug treatment (i.e. rapamycin) or modification of expression of proteins known to modulate its activity, and the direct role of raptor in insulin secretion is unclear. In this study, using siRNA, we investigated the role of raptor and rictor in insulin secretion and production in INS-1 cells and the possible cross talk between their respective complexes, mTORC1 and mTORC2. Reduced expression of raptor is associated with increased glucose-stimulated insulin secretion and intracellular insulin content. Downregulation of rictor expression leads to impaired insulin secretion without affecting insulin content and is able to correct the increased insulin secretion mediated by raptor siRNA. Using dominant-negative or constitutively active forms of Akt, we demonstrate that the effect of both raptor and rictor is mediated through alteration of Akt signaling. Our finding shed new light on the mechanism of control of insulin secretion and production by the mTOR, and they provide evidence for antagonistic effect of raptor and rictor on insulin secretion in response to glucose by modulating the activity of Akt, whereas only raptor is able to control insulin biosynthesis.

Context: For the last 10 yr, continuous glucose monitoring (CGM) has brought up new insights into the accuracy of blood glucose analysis., Objective: Our objective was to determine how islet graft function was able to influence the various components of dysglycemia after islet transplantation (IT)., Design and Setting: We conducted a single-arm open-labeled study with a 3-yr follow-up in a referral center (ClinicalTrial.gov identifiers NCT00446264 and NCT01123187)., Patients: Twenty-three consecutive patients with type 1 diabetes (14 islet alone, nine islet after kidney) received IT within 3 months using the Edmonton protocol., Intervention: INTERVENTION included 72-h CGM before and 3, 6, 9, 12, 24, and 36 months after transplantation., Main Outcome Measure: Graft function was estimated via β-score, a previously validated index (range 0-8) based on treatment requirements, C-peptide, blood glucose, and glycated hemoglobin., Results: At the 3-yr visit, graft function persisted in 19 patients (82%), and 10 (43%) remained insulin independent. Glycated hemoglobin decreased in the whole cohort from 8.3% (7.3-9.0%) at baseline to 6.7% (5.9-7.7%) at 3 yr [median (interquartile range), P < 0.01]. Mean glucose, glucose sd, and time spent with glycemia above 10 mmol/liter (hyperglycemia) and below 3 mmol/liter (hypoglycemia) were significantly lower after IT (P < 0.05 vs. baseline). The four CGM outcomes were related to β-score (P < 0.001). However, partial function (β-score >3) was sufficient to abrogate hypoglycemia; suboptimal function (β-score >5) was necessary to significantly improve mean glucose, glucose sd, and hyperglycemia; and optimal function (β score >7) was necessary to normalize them., Conclusion: The four components of dysglycemia were not equally affected by the degree of islet graft function, which could have important implications for future development of β-cell replacement. A β-score above 3 dramatically reduced the occurrence of hypoglycemia.

A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and β cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the β cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a β cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to β cell programming and diabetes pathophysiology., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., Methods: Paraffin sections of pancreases from 187 brain-deceased donors (HbA(1c) <6.5% [48 mmol/mol]) were used to genotype the TCF7L2 variant rs7903146 and evaluate its impact on islet morphology and alpha and beta cell subpopulations following immunostaining for glucagon and C-peptide. Following islet isolation, we investigated the correlation between TCF7L2 genotype and in vitro islet functional variables from our in-house pancreatic database., Results: TCF7L2 rs7903146 (T/T) was associated with reduced basal and glucose-stimulated insulin secretion in isolated human islets, and reduced islet density in whole pancreas. Morphological analysis demonstrated islet size was increased in T/T carriers. Furthermore, rs7903146 was associated with an increased glucagon/C-peptide ratio, especially in bigger islets., Conclusion/interpretation: The TCF7L2 variant rs7903146 risk allele is associated with impaired insulin secretion, reduction of total islet number and quantitative as well as qualitative morphological changes in human islets. Understanding how the TCF7L2 genotype modulates its activity and how TCF7L2 impacts the islet morphology may aid the design of new therapeutic approaches for the treatment of type 2 diabetes.

Zinc ions are essential for the formation of insulin crystals in pancreatic β cells, thereby contributing to packaging efficiency of stored insulin. Zinc fluxes are regulated through the SLC30A (zinc transporter, ZNT) family. Here, we investigated the effect of metabolic stress associated with the prediabetic state (zinc depletion, glucotoxicity, and lipotoxicity) on ZNT expression and human pancreatic islet function. Both zinc depletion and lipotoxicity (but not glucotoxicity) downregulated ZNT8 (SLC30A8) expression and altered the glucose-stimulated insulin secretion index (GSIS). ZNT8 overexpression in human islets protected them from the decrease in GSIS induced by tetrakis-(2-pyridylmethyl) ethylenediamine and palmitate but not from cell death. In addition, zinc supplementation decreased palmitate-induced human islet cell death without restoring GSIS. Altogether, we showed that ZNT8 expression responds to variation in zinc and lipid levels in human β cells, with repercussions on insulin secretion. Prospects for increasing ZNT8 expression and/or activity may prove beneficial in type 2 diabetes in humans.

The NR4A orphan nuclear receptors Nur77, Nurr1, and Nor1 exert multiple cellular and metabolic functions. These transcriptional regulators are activated in response to extracellular stresses, including lipotoxic fatty acids (FA) and proinflammatory cytokines. The contribution of NR4As to β-cell pathophysiology is, however, unknown. We have therefore examined the role of NR4As as downstream contributors to FA-induced β-cell dysfunctions. Human pancreatic islets and insulinoma β-cells were used to determine transcriptional programs elicited by NR4A, which were compared to those triggered by palmitate treatment. Functional studies evaluated the consequence of an increased NR4A expression on insulin biosynthesis and secretion and cell viability in insulinoma β-cells. FA and cytokine treatment increased NR4A expression in pancreatic β-cells, with Nur77 being most highly inducible in murine β-cells. Nur77, Nurr1, or Nor1 modulated common and distinct clusters of genes involved notably in cation homeostasis and insulin gene transcription. By altering zinc homeostasis, insulin gene transcription, and secretion, Nur77 was found to be a major transcriptional mediator of part of FA-induced β-cell dysfunctions. The repressive role of Nur77 in insulin gene regulation was tracked down to protein-protein interaction with FoxO1, a pivotal integrator of the insulin gene regulatory network. The present study identifies a member of the NR4A nuclear receptor subclass, Nur77/NR4A1, as a modulator of pancreatic β-cell biology. Together with its previously documented role in liver and muscle, its role in β-cells establishes Nur77 as an important integrator of glucose metabolism.

It has been shown that adult pancreatic ductal cells can dedifferentiate and act as pancreatic progenitors. Dedifferentiation of epithelial cells is often associated with the epithelial-mesenchymal transition (EMT). In this study, we investigated the occurrence of EMT in adult human exocrine pancreatic cells both in vitro and in vivo. Cells of exocrine fraction isolated from the pancreas of brain-dead donors were first cultured in suspension for eight days. This led to the formation of spheroids, composed of a principal population of cells with duct-like phenotype. When cultivated in tissue culture-treated flasks, spheroid cells exhibited a proliferative capacity and coexpressed epithelial (cytokeratin7 and cytokeratin19) and mesenchymal (vimentin and alpha-smooth muscle actin) markers as well as marker of progenitor pancreatic cells (pancreatic duodenal homeobox factor-1) and surface markers of mesenchymal stem cells. The switch from E-cadherin to N-cadherin associated with Snail1 expression suggested that these cells underwent EMT. In addition, we showed coexpression of epithelial and mesenchymal markers in ductal cells of one normal adult pancreas and three type 2 diabetic pancreases. Some of the vimentin-positive cells were found to coexpress glucagon or amylase. These results point to the occurrence of EMT, which may take place on dedifferentiation of ductal cells during the regeneration or renewal of human pancreatic tissues.