Pharmaceutical targeting of the cannabinoid type 1 receptor impacts the crosstalk between immune cells and islets to reduce insulitis in humans.

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-1ß, 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 .

Primary Graft Function and 5 Year Insulin Independence After Pancreas and Islet Transplantation for Type 1 Diabetes: A Retrospective Parallel Cohort Study.

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.

The Map3k12 (Dlk)/JNK3 signaling pathway is required for pancreatic beta-cell proliferation during postnatal development.

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.

Characterization of one anastomosis gastric bypass and impact of biliary and common limbs on bile acid and postprandial glucose metabolism in a minipig model.

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.

Pharmacological approach to understanding the control of insulin secretion in human islets.

AIMS: To understand better the control of insulin secretion by human ß 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 KATP 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 KATP channels: Kv or BK channels (tetraethylammonium), TASK-1 channels (ML-365) and SK4 channels (TRAM-34). Opening KATP 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 KATP channel-dependent triggering pathway and a metabolic amplifying pathway, human and rodent ß cells were found to display more similarities than differences in the control of insulin secretion.

Comparison of fluid balance and hemodynamic and metabolic effects of sodium lactate versus sodium bicarbonate versus 0.9% NaCl in porcine endotoxic shock: a randomized, open-label, controlled study.

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/m2 at 300 min versus the NC (207 (119-272) mL/min/m2, p = 0.01) and the SB (278, (211-315) mL/min/m2, p = 0.03) groups). Oxygenation markers (arterial oxygen partial pressure (PaO2)/inspired oxygen fraction (FiO2), mixed venous oxygen saturation (SvO2), and venoarterial carbon dioxide tension difference (Pv-aCO2) 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.

Hypertonic sodium lactate improves fluid balance and hemodynamics in porcine endotoxic shock.

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.

Coxsackievirus B4 can infect human pancreas ductal cells and persist in ductal-like cell cultures which results in inhibition of Pdx1 expression and disturbed formation of islet-like cell aggregates.

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 ß-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 ß-cells.

Effects of subtotal pancreatectomy and long-term glucose and lipid overload on insulin secretion and glucose homeostasis in minipigs.

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.

The NERP-4-SNAT2 axis regulates pancreatic ß-cell maintenance and function.

Insulin secretion from pancreatic ß 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 ß cells. The granin protein VGF has dual roles in ß cells: regulating secretory granule formation and functioning as a multiple peptide precursor. A VGF-derived peptide, neuroendocrine regulatory peptide-4 (NERP-4), increases Ca2+ influx in the pancreata of transgenic mice expressing apoaequorin, a Ca2+-induced bioluminescent protein complex. NERP-4 enhances glucose-stimulated insulin secretion from isolated human and mouse islets and ß-cell-derived MIN6-K8 cells. NERP-4 administration reverses the impairment of ß-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 ß cells and stimulating insulin secretion. SNAT2 deletion and inhibition abolish the protective effects of NERP-4 on ß-cell maintenance. These findings demonstrate a novel autocrine mechanism of ß-cell maintenance and function that is mediated by the peptide-amino acid transporter axis.

The challenge of HLA donor specific antibodies in the management of pancreatic islet transplantation: an illustrative case-series.

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 ß-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.

Innovative transdermal delivery of insulin using gelatin methacrylate-based microneedle patches in mice and mini-pigs.

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 MoS2 nanosheets as a photothermal component into MN hydrogels results in MNs featuring on-demand release properties. An optimized MoS2-MN array patch formed using a hydrogel solution containing 500 µg mL-1 of MoS2 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 MoS2-MN patches can be used for loading and releasing insulin for therapeutic purposes. Indeed, transdermal administration of insulin loaded into MoS2-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.

Pancreatic islet transplantation under the kidney capsule of mice: model of refinement for molecular and ex-vivo graft analysis.

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.

Effects of proprotein convertase subtilisin kexin type 9 modulation in human pancreatic beta cells function.

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-ßH1 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-ßH1 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-ßH1 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.

5'-AZA induces Ngn3 expression and endocrine differentiation in the PANC-1 human ductal cell line.

Neurogenin 3 is necessary for endocrine cell development in the embryonic pancreas and has been shown to induce transdifferentiation duct cells from adult pancreas toward a neuro-endocrine phenotype. Here we discovered that the demethylating agent 5'-Azadeoxycytidine (AZA) induced Ngn3 expression and endocrine differentiation from the PANC-1 human ductal cell line. The expression of markers specific to mature islet cells, i.e., glucagon and somatostatin, was also observed. In addition, we demonstrated that growth factors (betacellulin and soluble factors released during pancreas embryogenesis) increased the level of maturation. Our studies revealed that the PANC-1 model system may provide a basis for elucidating the ductal/endocrine differentiation.

Efficient gene delivery and silencing of mouse and human pancreatic islets.

BACKGROUND: In view of the importance of beta cells in glucose homeostasis and the profound repercussions of beta cell pathology on human health, the acquisition of tools to study pancreatic islet function is essential for the design of alternative novel therapies for diabetes. One promising approach toward this goal involves the modification of gene expression profile of beta cells. RESULTS: This study describes a new method of gene and siRNA delivery into human pancreatic islets by microporation technology. We demonstrated that mild islet distention with accutase greatly enhanced the transfection efficiency without compromising in vitro function (secretion, apoptosis and viability). As an example, the recently identified gene involved in type 2 diabetes, ZnT8, can be over-expressed or silenced by RNA interference using this technology. Microporation can also be used on rodent islets. CONCLUSIONS: Taken together, our results demonstrate that microporation technology can be used to modify gene expression in whole rodent and human islets without altering their in vitro function and will be key to the elucidation of the factors responsible for proper islet function.

Coxsackievirus-B4 Infection Can Induce the Expression of Human Endogenous Retrovirus W in Primary Cells.

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.

Interindividual Heterogeneity of SGLT2 Expression and Function in Human Pancreatic Islets.

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.

PCSK9 is expressed in pancreatic delta-cells and does not alter insulin secretion.

PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a proprotein convertase that plays a key role in cholesterol homeostasis by decreasing hepatic low-density lipoprotein receptor (LDLR) protein expression. Here, we investigated the expression and the function of PCSK9 in pancreatic islets. Immunohistochemistry analysis showed that PCSK9 co-localized specifically with somatostatin in human pancreatic delta-cells, with no expression in alpha- and beta-cells. PCSK9 seems not to be secreted by mouse isolated islets maintained in culture. Pcsk9-deficiency led to a 200% increase in LDLR protein content in mouse isolated islets, mainly in beta-cells. Conversely, incubation of islets with recombinant PCSK9 almost abolished LDLR expression. However, Pcsk9-deficiency did not alter cholesterol content nor glucose-stimulated insulin secretion in mouse islets. Finally, invivo glucose tolerance was similar in Pcsk9(+/+) and Pcsk9(-/-) mice under basal conditions and following streptozotocin treatment. These results suggest, at least in mice, that PCSK9 does not alter insulin secretion.

Single Low Dose of Human Recombinant Antithrombin (ATryn) has no Impact on Endotoxin-Induced Disseminated Intravascular Coagulation: An Experimental Randomized Open Label Controlled Study.

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.