Inhibition of Toll-like Receptor 4 Using Small Molecule, TAK-242, Protects Islets from Innate Immune Responses.

Islet transplantation is a therapeutic option to replace ß-cell mass lost during type 1 or type 3c diabetes. Innate immune responses, particularly the instant blood-mediated inflammatory reaction and activation of monocytes, play a major role in the loss of transplanted islet tissue. In this study, we aimed to investigate the inhibition of toll-like receptor 4 (TLR4) on innate inflammatory responses. We first demonstrate a significant loss of graft function shortly after transplant through the assessment of miR-375 and miR-200c in plasma as biomarkers. Using in vitro models, we investigate how targeting TLR4 mitigates islet damage and immune cell activation during the peritransplant period. The results of this study support the application of TAK-242 as a therapeutic agent to reduce inflammatory and innate immune responses to islets immediately following transplantation into the hepatic portal vein. Therefore, TLR4 may serve as a target to improve islet transplant outcomes in the future.

Clinical and biological significance of circulating miRNAs in chronic pancreatitis patients undergoing total pancreatectomy with islet autotransplantation.

BACKGROUND: Specific microRNAs (miRNAs) were elevated in chronic pancreatitis (CP) patients during islet infusion after total pancreatectomy (TPIAT). We aimed to identify circulating miRNA signatures of pancreatic damage, predict miRNA-mRNA networks to identify potential links to CP pathogenesis and identify islet isolation and transplantation functional outcomes. METHODS: Small RNA sequencing was performed to identify distinct circulating miRNA signatures in CP. Plasma miRNAs were measured using miRCURY LNA SYBR green quantitative real-time polymerase chain reaction assays. Correlation analyses were performed using R software. The miRNA target and disease interactions were determined using miRNet and the miRNA enrichment and annotation tool. RESULTS: Alterations were found in circulating miRNAs in CP patients compared to healthy controls. Further studies were conducted on 12 circulating miRNAs enriched in the pancreas, other tissues and other diseases including cancer and fibrosis. Approximately 2888 mRNAs in the pancreas were their targets, demonstrating interactions with 76 small molecules. Three miRNAs exhibited interactions with morphine and five exhibited interactions with glucose. The miRNA panel targeted 22 genes associated with pancreatitis. The islet-specific, acinar cell-specific and liver-specific miRNAs were elevated at 6 h after islet infusion and returned to baseline levels 3 months after TPIAT. Circulating levels of miRNAs returned to pre-transplant levels 1-year post-transplant. Circulating miRNAs measured before and 6 h after islet infusion were directly or inversely associated with metabolic outcomes at 3 and 6 months post-transplant. CONCLUSIONS: miRNAs may contribute to CP pathogenesis, and elevated circulating levels may be specific to pancreatic inflammation and fibrosis, warranting further investigation.

Calcineurin/NFATc2 and PI3K/AKT signaling maintains ß-cell identity and function during metabolic and inflammatory stress.

Pancreatic islets respond to metabolic and inflammatory stress by producing hormones and other factors that induce adaptive cellular and systemic responses. Here we show that intracellular Ca2+ ([Ca2+]i) and ROS signals generated by high glucose and cytokine-induced ER stress activate calcineurin (CN)/NFATc2 and PI3K/AKT to maintain ß-cell identity and function. This was attributed in part by direct induction of the endocrine differentiation gene RFX6 and suppression of several ß-cell "disallowed" genes, including MCT1. CN/NFATc2 targeted p300 and HDAC1 to RFX6 and MCT1 promoters to induce and suppress gene transcription, respectively. In contrast, prolonged exposure to stress, hyperstimulated [Ca2+]i, or perturbation of CN/NFATc2 resulted in downregulation of RFX6 and induction of MCT1. These findings reveal that CN/NFATc2 and PI3K/AKT maintain ß-cell function during acute stress, but ß-cells dedifferentiate to a dysfunctional state upon loss or exhaustion of Ca2+/CN/NFATc2 signaling. They further demonstrate the utility of targeting CN/NFATc2 to restore ß-cell function.

Role of Exosomes in Islet Transplantation.

Exosomes are known for their ability to transport nucleic acid, lipid, and protein molecules, which allows for communication between cells and tissues. The cargo of the exosomes can have a variety of effects on a wide range of targets to mediate biological function. Pancreatic islet transplantation is a minimally invasive cell replacement therapy to prevent or reverse diabetes mellitus and is currently performed in patients with uncontrolled type 1 diabetes or chronic pancreatitis. Exosomes have become a focus in the field of islet transplantation for the study of diagnostic markers of islet cell viability and function. A growing list of miRNAs identified from exosomes collected during the process of isolating islets can be used as diagnostic biomarkers of islet stress and damage, leading to a better understanding of critical steps of the isolation procedure that can be improved to increase islet yield and quality. Exosomes have also been implicated as a possible contributor to islet graft rejection following transplantation, as they carry donor major histocompatibility complex molecules, which are then processed by recipient antigen-presenting cells and sensed by the recipient immune cells. Exosomes may find their way into the therapeutic realm of islet transplantation, as exosomes isolated from mesenchymal stem cells have shown promising results in early studies that have seen increased viability and functionality of isolated and grafted islets in vitro as well as in vivo. With the study of exosomes still in its infancy, continued research on the role of exosomes in islet transplantation will be paramount to understanding beta cell regeneration and improving long-term graft function.

Withaferin A inhibits lymphocyte proliferation, dendritic cell maturation in vitro and prolongs islet allograft survival.

The immunosuppressive regimen for clinical allogeneic islet transplantation uses beta cell-toxic compounds such as tacrolimus that cause islet graft loss. Previously we reported that the plant-derived steroidal lactone Withaferin A (WA) can protect islet grafts by inhibiting nuclear factor-kappa B (NF-κB). Since the NF-κB signaling pathway is essential for T-cell activation, we hypothesized that long-term WA administration may also provide an immunosuppressive effect. Treatment of BALB/c donor islets and C57BL/6N recipients with WA alone resulted in 80% islet graft long-term survival vs. 40% in low-dose FK506-treated mice. In vitro, WA significantly blocked mouse and human T-cell proliferation by CD3/CD28 bead stimulation and in mixed lymphocyte reaction assay. Treatment of immature dendritic cells with WA prevented their maturation in response to inflammatory stimuli, as seen by decreased expression of CD83 and human leukocyte antigen-DR isotype. Exosomes released by islets treated with WA contained significantly fewer proinflammatory molecules interleukin-6, interleukin-8, monocyte chemoattractant protein-1, interferon-gamma-induced protein-10, inducible nitric oxide synthase, and cyclooxygenase-2. In conclusion, WA treatment not only reduced inflammation but also prolonged allograft survival, possibly through suppression of dendritic cell maturation and T-cell proliferation. WA has the potential to inhibit both the innate and adaptive immune response to prolong allograft survival.

Circulating miRNA in Patients Undergoing Total Pancreatectomy and Islet Autotransplantation.

Circulating microRNAs (miRNAs) can be biomarkers for diagnosis and progression of several pathophysiological conditions. In a cohort undergoing total pancreatectomy with islet autotransplantation (TPIAT) from the multicenter Prospective Observational Study of TPIAT (POST), we investigated associations between a panel of circulating miRNAs (hsa-miR-375, hsa-miR-29b-3p, hsa-miR-148a-3p, hsa-miR-216a-5p, hsa-miR-320d, hsa-miR-200c, hsa-miR-125b, hsa-miR-7-5p, hsa-miR-221-3p, hsa-miR-122-5p) and patient, disease and islet-isolation characteristics. Plasma samples (n = 139) were collected before TPIAT and miRNA levels were measured by RTPCR. Disease duration, prior surgery, and pre-surgical diabetes were not associated with circulating miRNAs. Levels of hsa-miR-29b-3p (P = 0.03), hsa-miR-148a-3p (P = 0.04) and hsa-miR-221-3p (P = 0.01) were lower in those with genetic risk factors. Levels of hsa-miR-148a-3p (P = 0.04) and hsa-miR-7-5p (P = 0.04) were elevated in toxic/metabolic disease. Participants with exocrine insufficiency had lower hsa-miR-29b-3p, hsa-miR-148a-3p, hsa-miR-320d, hsa-miR-221-3p (P < 0.01) and hsa-miR-375, hsa-miR-200c-3p, and hsa-miR-125b-5p (P < 0.05). Four miRNAs were associated with fasting C-peptide before TPIAT (hsa-miR-29b-3p, r = 0.18; hsa-miR-148a-3p, r = 0.21; hsa-miR-320d, r = 0.19; and hsa-miR-221-3p, r = 0.21; all P < 0.05), while hsa-miR-29b-3p was inversely associated with post-isolation islet equivalents/kg and islet number/kg (r = -0.20, P = 0.02). Also, hsa-miR-200c (r = 0.18, P = 0.03) and hsa-miR-221-3p (r = 0.19, P = 0.03) were associated with islet graft tissue volume. Further investigation is needed to determine the predictive potential of these miRNAs for assessing islet autotransplant outcomes.

Outcomes of Islet Autotransplantation in Chronic Pancreatitis Patients with Complete Acinar Atrophy.

Total pancreatectomy with islet autotransplantation (TPIAT) is a promising treatment for refractory chronic pancreatitis (CP). Pathological features of CP include progressive fibrosis in pancreas parenchyma, atrophy, and/or ductal occlusion. Complete acinar atrophy (CAA) caused by chronic fibrosis and necroinflammation results in exocrine sufficiency and may influence islet isolation characteristics during TPIAT. In this analysis of patients who underwent TPIAT at our center, we compared transplant outcomes among those with CAA (n = 5) vs non-acinar atrophy (NAA; matching controls, n = 36). Data were analyzed using one-way analysis of variance with Bonferroni post hoc test or Student's t test. Pancreas digestion was longer in CAA than in NAA cases (18.6 vs 14.6 min) despite a lower pancreas weight (55.2 vs 91.2 g). Obtained tissue volume was 1.0 ml in the CAA group and 12.1 ml in the NAA group. Both groups had similar islet viability (96%) and islet dose (CAA, 3,391 IEQ/kg; NAA, 4141.1 IEQ/kg). During islet infusion, serum cytokine (IL-6, IL-8, and MCP-1) levels and plasma hsa-miR-375 levels were lower in the CAA group than in the NAA group, but not significantly. Serum tumor necrosis factor α levels at 3 h after infusion were significantly higher in CAA group than in NAA group. After TPIAT, the metabolic outcomes of the CAA group were comparable with that of the NAA group. Narcotics usage decreased significantly over 24 months in both groups, with the CAA group reporting being pain free at 12 months. Complete atrophy of acinar cells of pancreas did not significantly impact islet yield or endocrine function after TPIAT.

Grafting Islets to a Dissected Peritoneal Pouch to Improve Transplant Survival and Function.

BACKGROUND: Although the liver is the primary site for clinical islet transplantation, it poses several restrictions, especially limited tissue volume due to portal vein pressure. We evaluated the preperitoneal space as an extrahepatic islet transplant site to deliver high tissue volumes and sustain long-term graft function. METHODS: A peritoneal pouch was formed by dissecting the parietal peritoneum from the transversalis fascia of mice. Syngeneic C57BL/6 donor islets were transplanted into the peritoneal pouch of diabetic mouse recipients. Blood glucose was monitored for islet function, and miR-375 was analyzed for islet damage. Islet graft morphology and vascularization were evaluated by immunohistochemistry. [F] fluoro-D-glucose positron emission tomography/computed tomography was used to image islet grafts. RESULTS: Transplantation of 300 syngeneic islets into the peritoneal pouch of recipients reversed hyperglycemia for >60 days. Serum miR-375 was significantly lower in the peritoneal pouch group than in the peritoneal cavity group. Peritoneal pouch islet grafts showed high neovascularization and sustained insulin and glucagon expression up to 80 days posttransplantation. A peritoneal pouch graft with high tissue volume (1000 islets) could be visualized by positron emission tomography/computed tomography imaging. Human islets transplanted into the peritoneal pouch of diabetic nude mice also reversed hyperglycemia successfully. CONCLUSIONS: Islets transplanted into a dissected peritoneal pouch show high efficiency to reverse diabetes and sustain islet graft function. The preperitoneal site has the advantages of capacity for high tissue volume, enriched revascularization and minimal inflammatory damage. It can also serve as an extrahepatic site for transplanting large volume of islets necessitated in islet autotransplantation.

Impact of microbial contamination of the islet product during total pancreatectomy with islet autotransplantation.

BACKGROUND: The combined use of interleukin-1ß and tumor necrosis factor-α blockers in the peritransplant period has improved outcomes of total pancreatectomy with islet autotransplantation (TPIAT). However, these drugs may suppress the immune system, resulting in severe infection. METHODS: We retrospectively investigated the impact of microbial-contaminated islet product on posttransplant complications and metabolic outcomes of TPIAT patients receiving the IL-1ß and TNF-blockade treatment at our center. RESULTS: Among 108 TPIAT patients, 37 patients (34%) received contaminated products. Preoperative stent treatment and fibrosis score were independent risk factors for the contamination. There were no significant differences between the contaminated and noncontaminated product groups in posttransplant infectious complication rate, length of hospitalization, or readmission rate. However, islet equivalents (P < .0001) and insulin independence rate (P = .036) at 6 months were significantly lower for patients receiving contaminated product. CONCLUSIONS: These results suggest that combined anti-inflammatory drug use is safe and well tolerated in TPIAT patients who receive contaminated islet product and does not increase the rate of infectious complications; however, contaminated islet product is associated with poor metabolic outcomes.

MicroRNA Signatures as Future Biomarkers for Diagnosis of Diabetes States.

Diabetes results from the inability of pancreatic islets to maintain blood glucose concentrations within a normal physiological range. Clinical features are usually not observed until islets begin to fail and irreversible damage has occurred. Diabetes is generally diagnosed based on elevated glucose, which does not distinguish between type 1 and 2 diabetes. Thus, new diagnostic approaches are needed to detect different modes of diabetes before manifestation of disease. During prediabetes (pre-DM), islets undergo stress and release micro (mi) RNAs. Here, we review studies that have measured and tracked miRNAs in the blood for those with recent-onset or longstanding type 1 diabetes, obesity, pre-diabetes, type 2 diabetes, and gestational diabetes. We summarize the findings on miRNA signatures with the potential to stage progression of different modes of diabetes. Advances in identifying selective biomarker signatures may aid in early detection and classification of diabetic conditions and treatments to prevent and reverse diabetes.

Differential expression and release of exosomal miRNAs by human islets under inflammatory and hypoxic stress.

AIMS/HYPOTHESIS: Pancreatic islets produce non-coding microRNAs (miRNAs) that regulate islet cell function and survival. Our earlier investigations revealed that human islets undergo significant damage due to various types of stresses following transplantation and release miRNAs. Here, we sought to identify and validate exosomal miRNAs (exo-miRNAs) produced by human islets under conditions of cellular stress, preceding loss of cell function and death. We also aimed to identify islet stress signalling pathways targeted by exo-miRNAs to elucidate potential regulatory roles in islet cell stress. METHODS: Human islets were subjected to proinflammatory cytokine and hypoxic cell stress and miRNA from exosomes was isolated for RNA sequencing and analysis. Stress-induced exo-miRNAs were evaluated for kinetics of expression and release by intact islets for up to 48 h exposure to cytokines and hypoxia. A subset of stress-induced exo-miRNAs were assessed for recovery and detection as biomarkers of islet cell stress in a diabetic nude mouse xenotransplant model and in patients undergoing total pancreatectomy with islet auto-transplantation (TPIAT). Genes and signalling pathways targeted by stress-induced exo-miRNAs were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and direct interactions of miRNAs with downstream signalling targets were validated in human islet cells using the miRNA Tests for Read Analysis and Prediction (MirTrap) system. RESULTS: Global exo-miRNA sequencing revealed that 879 miRNA species were released from human islets and 190 islet exo-miRNAs were differentially expressed in response to proinflammatory cytokines, hypoxia or both. Release of exo-miRNAs hsa-miR-29b-3p and hsa-miR-216a-5p was detected within 6 h of exposure to cytokines and hypoxia. The remaining subset of stress-induced exo-miRNAs, including hsa-miR-148a-3p and islet cell damage marker hsa-miR-375, showed delayed release at 24-48 h, correlating with apoptosis and cell death. Stress and damage exo-miRNAs were significantly elevated in the circulation in human-to-mouse xenotransplant models and in human transplant recipients. Elevated blood exo-miRNAs negatively correlated with post-transplant islet function based on comparisons of stress and damage exo-miRNA indices with Secretory Unit of Islet Transplant Objects (SUITO) indices. KEGG analysis and further validation of exo-miRNA targets by MirTrap analysis revealed significant enrichment of islet mRNAs involved in phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signalling pathways. CONCLUSIONS/INTERPRETATION: The study identifies exo-miRNAs differentially expressed and released by islets in response to damage and stress. These exo-miRNAs could serve as potential biomarkers for assessing islet damage and predicting outcomes in islet transplantation. Notably, exo-miRNAs 29b-3p and 216a-5p could be detected in islets prior to damage-released miRNAs and indicators of cellular apoptosis and death. Thus, these stress-induced exo-miRNAs may have potential diagnostic value for detecting early islet stress prior to progressive loss of islet cell mass and function. Further investigations are warranted to investigate the utility of these exo-miRNAs as early indicators of islet cell stress during prediabetic conditions.

Profiling Gene Programs in the Blood During Liver Regeneration in Living Liver Donors.

The human liver's capacity to rapidly regenerate to a full-sized functional organ after resection has allowed successful outcomes for living donor liver transplantation (LDLT) procedures. However, the ability to detect and track physiological changes occurring during liver regeneration after resection and throughout the restoration process is still lacking. We performed a comprehensive whole-transcriptome RNA sequencing analysis of liver and circulating blood tissue from 12 healthy LDLT donors to define biomarker signatures for monitoring physiological activities during liver regeneration at 14 time points for up to a 1-year procedural follow-up. LDLT donor liver tissue differentially expressed 1238 coding and noncoding genes after resection, and an additional 1260 genes were selectively regulated after LDLT. A total of 15,011 RNA transcript species were identified in the blood in response to liver resection. The transcripts most highly regulated were sequentially expressed within 3 distinct peaks that correlated with sets of functional genes involved in the induction of liver resection-specific innate immune response (peak 1), activation of the complement system (peak 2), and platelet activation and erythropoiesis (peak 3). Each peak corresponded with progressive phases of extracellular matrix degradation, remodeling, and organization during liver restoration. These processes could be tracked by distinct molecular signatures of up-regulated and down-regulated gene profiles in the blood during phases of liver repair and regeneration. In conclusion, the results establish temporal and dynamic transcriptional patterns of gene expression following surgical liver resection that can be detected in the blood and potentially used as biomarker signatures for monitoring phases of liver regeneration.

Expression of Gastrin Family Peptides in Pancreatic Islets and Their Role in ß-Cell Function and Survival.

OBJECTIVES: Modulation of cholecystokinin (CCK) receptors has been shown to influence pancreatic endocrine function. METHODS: We assessed the impact of the CCKA and CCKB receptor modulators, (pGlu-Gln)-CCK-8 and gastrin-17, respectively, on ß-cell secretory function, proliferation and apoptosis and glucose tolerance, and investigating alterations of CCK and gastrin islet expression in diabetes. RESULTS: Initially, the presence of CCK and gastrin, and expression of their receptors were evidenced in ß-cell lines and mouse islets. (pGlu-Gln)-CCK-8 and gastrin-17 stimulated insulin secretion from BRIN-BD11 and 1.1B4 ß-cells, associated with no effect on membrane potential or [Ca]i. Only (pGlu-Gln)-CCK-8 possessed insulin secretory actions in isolated islets. In agreement, (pGlu-Gln)-CCK-8 improved glucose disposal and glucose-induced insulin release in mice. In addition, (pGlu-Gln)-CCK-8 evoked clear satiety effects. Interestingly, islet colocalization of CCK with glucagon was elevated in streptozotocin- and hydrocortisone-induced diabetic mice, whereas gastrin coexpression in α cells was reduced. In contrast, gastrin colocalization within ß-cells was higher in diabetic mice, while CCK coexpression with insulin was decreased in insulin-deficient mice. (pGlu-Gln)-CCK-8 and gastrin-17 also augmented human and rodent ß-cell proliferation and offered protection against streptozotocin-induced ß-cell cytotoxicity. CONCLUSIONS: We highlight the direct involvement of CCKA and CCKB receptors in pancreatic ß-cell function and survival.

Anti-diabetic actions of esculentin-2CHa(1-30) and its stable analogues in a diet-induced model of obesity-diabetes.

Actions of esculentin-2CHa(1-30) (GFSSIFRGVAKFASKGLGKDLAKLGVDLVA) and its analogues, ([D-Arg7, D-Lys15, D-Lys23]-esculentin-2CHa(1-30) and [Lys15-octanoate]-esculentin-2CHa(1-30), were evaluated in high-fat fed NIH Swiss mice with impaired glucose tolerance and insulin resistance. Twice-daily i.p. administration of the esculentin-2CHa(1-30) peptides (75 nmol/kg body weight) or exendin-4 (25 nmol/kg) for 28 days reduced body weight, without altering cumulative energy intake. All peptides reduced blood glucose levels by 6-12 mmol/l concomitant with lower plasma insulin levels, with significance evident from day 6. All peptides improved glucose tolerance, insulin sensitivity, blood glucose profile over 24 h and decreased HbA1c to a similar extent as exendin-4. The peptides also reduced high fat diet-induced increases in plasma GLP-1 and glucagon. None of the peptides altered bone mineral density/content or lean mass but decreased fat mass. Islets isolated from peptide-treated mice exhibited improved glucose-, alanine- and GLP-1-stimulated insulin secretion. Islet morphometric analyses revealed that exendin-4 and the esculentin-2CHa(1-30) peptides significantly reduced islet, beta and alpha cell areas compared to high-fat controls. Esculentin-2CHa(1-30) peptides markedly reduced high fat diet-induced increase in beta cell proliferation and apoptosis. Peptide treatments had beneficial effects on expression of islet genes (Ins1, Slc2a2, Pdx1) and skeletal muscle genes involved in insulin action (Slc2a4, Pdk1, Irs1, Akt1). High-fat diet significantly increased LDL cholesterol which was reduced by the acylated esculentin-2CHa(1-30) analogue. Peptide treatments did not alter circulating concentrations of amylase and marker enzymes of liver function, indicating a lack of toxicity. These data indicate that esculentin-2CHa(1-30) and its analogues may be useful for improvement of blood glucose control and weight loss in type 2 diabetes.

Locally produced xenin and the neurotensinergic system in pancreatic islet function and ß-cell survival.

Modulation of neuropeptide receptors is important for pancreatic ß-cell function. Here, islet distribution and effects of the neurotensin (NT) receptor modulators, xenin and NT, was examined. Xenin, but not NT, significantly improved glucose disposal and insulin secretion, in mice. However, both peptides stimulated insulin secretion from rodent ß-cells at 5.6 mm glucose, with xenin having similar insulinotropic actions at 16.7 mm glucose. In contrast, NT inhibited glucose-induced insulin secretion. Similar observations were made in human 1.1B4 ß-cells and isolated mouse islets. Interestingly, similar xenin levels were recorded in pancreatic and small intestinal tissue. Arginine and glucose stimulated xenin release from islets. Streptozotocin treatment decreased and hydrocortisone treatment increased ß-cell mass in mice. Xenin co-localisation with glucagon was increased by streptozotocin, but unaltered in hydrocortisone mice. This corresponded to elevated plasma xenin levels in streptozotocin mice. In addition, co-localisation of xenin with insulin was increased by hydrocortisone, and decreased by streptozotocin. Further in vitro investigations revealed that xenin and NT protected ß-cells against streptozotocin-induced cytotoxicity. Xenin augmented rodent and human ß-cell proliferation, whereas NT displayed proliferative actions only in human ß-cells. These data highlight the involvement of NT signalling pathways for the possible modulation of ß-cell function.

Differential expression of glucagon-like peptide-2 (GLP-2) is involved in pancreatic islet cell adaptations to stress and beta-cell survival.

Recent studies have confirmed that locally released proglucagon derived gene products, other than glucagon, have a major influence on pancreatic endocrine function. We assessed the impact of glucagon-like peptide-2 (GLP-2) on beta-cell secretory function, proliferation and apoptosis, as well as glucose tolerance, feeding behaviour and islet adaptions to chemically-induced insulin deficiency and resistance. The GLP-2 receptor was evidenced on cultured rodent and human beta-cells, rodent alpha-cells and isolated mouse islets. GLP-2 had no effect on insulin secretion from beta-cells, or isolated mouse islets. In vivo, GLP-2 administration significantly (P<0.05 to P<0.01) decreased food intake in mice. Conversely, GLP-2 had no discernible effects on glucose disposal or insulin secretion. As expected, streptozotocin treatment decreased and hydrocortisone increased beta-cell mass in mice. GLP-2 was visualised in mouse islets and intestinal L-cells. Islet GLP-2 co-localisation with glucagon was significantly decreased (P<0.01) by both streptozotocin and hydrocortisone. In contrast, both interventions increased (P<0.05) co-localisation of GLP-2 with somatostatin. Interestingly, GLP-2 positive cells were reduced (P<0.05) in the intestines of streptozotocin, but not hydrocortisone, treated mice. Further in vitro investigations revealed that GLP-2 protected rodent and human 1.1B4 beta-cells against streptozotocin induced DNA damage. Furthermore, GLP-2 augmented (P<0.05) BRIN BD11 beta-cell proliferation, but was less efficacious in 1.1B4 cells. These data highlight the involvement of GLP-2 receptor signalling in the adaptations to pancreatic islet cell stress.

Esculentin-2CHa(1-30) and its analogues: stability and mechanisms of insulinotropic action.

The insulin-releasing effects, cellular mechanisms of action and anti-hyperglycaemic activity of 10 analogues of esculentin-2CHa lacking the cyclic C-terminal domain (CKISKQC) were evaluated. Analogues of the truncated peptide, esculentin-2CHa(1-30), were designed for plasma enzyme resistance and increased biological activity. Effects of those analogues on insulin release, cell membrane integrity, membrane potential, intracellular Ca2+ and cAMP levels were determined using clonal BRIN-BD11 cells. Their acute effects on glucose tolerance were investigated using NIH Swiss mice. d-Amino acid substitutions at positions 7(Arg), 15(Lys) and 23(Lys) and fatty acid (l-octanoate) attachment to Lys at position 15 of esculentin-2CHa(1-30) conveyed resistance to plasma enzyme degradation whilst preserving insulin-releasing activity. Analogues, [d-Arg7,d-Lys15,d-Lys23]-esculentin-2CHa(1-30) and Lys15-octanoate-esculentin-2CHa(1-30), exhibiting most promising profiles and with confirmed effects on both human insulin-secreting cells and primary mouse islets were selected for further analysis. Using chemical inhibition of adenylate cyclase, protein kinase C or phospholipase C pathways, involvement of PLC/PKC-mediated insulin secretion was confirmed similar to that of CCK-8. Diazoxide, verapamil and Ca2+ omission inhibited insulin secretion induced by the esculentin-2CHa(1-30) analogues suggesting an action on KATP and Ca2+ channels also. Consistent with this, the analogues depolarised the plasma membrane and increased intracellular Ca2+ Evaluation with fluorescent-labelled esculentin-2CHa(1-30) indicated membrane action, with internalisation; however, patch-clamp experiments suggested that depolarisation was not due to the direct inhibition of KATP channels. Acute administration of either analogue to NIH Swiss mice improved glucose tolerance and enhanced insulin release similar to that observed with GLP-1. These data suggest that multi-acting analogues of esculentin-2CHa(1-30) may prove useful for glycaemic control in obesity-diabetes.

Influence of neuropeptide Y and pancreatic polypeptide on islet function and beta-cell survival.

BACKGROUND: In the present study we assessed the impact of neuropeptide Y receptor (NPYR) modulators, neuropeptide Y (NPY) and pancreatic polypeptide (PP), on islet function and beta-cell survival. METHODS: The effects of NPY and PP on beta-cell function were examined in BRIN BD11 and 1.1B4 beta-cells, as well as isolated mouse islets. Involvement of both peptides in pancreatic islet adaptations to streptozotocin and hydrocortisone, as well as effects on beta-cell proliferation and apoptosis was also evaluated. RESULTS: Neither NPY nor PP affected in vivo glucose disposal or insulin secretion in mice. However, both peptides inhibited (p<0.05 to p<0.001) glucose stimulated insulin secretion from rat and human beta-cells. NPY exerted similar insulinostatic effects in isolated mouse islets. NPY and PP inhibited alanine-induced changes in BRIN BD11 cell membrane potential and (Ca2+)i. Streptozotocin treatment decreased and hydrocortisone treatment increased beta-cell mass in mice. In addition, streptozotocin, but not hydrocortisone, increased PP cell area. Streptozotocin also shifted the normal co-localisation of NPY with PP, towards more pronounced co-expression with somatostatin in delta-cells. Both streptozotocin and hydrocortisone increased pancreatic exocrine expression of NPY. More detailed in vitro investigations revealed that NPY, but not PP, augmented (p<0.01) BRIN BD11 beta-cell proliferation. In addition, both peptides exerted protective effects against streptozotocin-induced DNA damage in beta-cells. CONCLUSION: These data emphasise the involvement of PP, and particularly NPY, in the regulation of beta-cell mass and function. GENERAL SIGNIFICANCE: Modulation of PP and NPY signalling is suitable for further evaluation and possible clinical development for the treatment of diabetes.

Implanting 1.1B4 human ß-cell pseudoislets improves glycaemic control in diabetic severe combined immune deficient mice.

AIM: To investigate the potential of implanting pseudoislets formed from human insulin-releasing ß-cell lines as an alternative to islet transplantation. METHODS: In this study, the anti-diabetic potential of novel human insulin releasing 1.1B4 ß-cells was evaluated by implanting the cells, either as free cell suspensions, or as three-dimensional pseudoislets, into the subscapular region of severe combined immune deficient mice rendered diabetic by single high-dose administration of streptozotocin. Metabolic parameters including food and fluid intake, bodyweight and blood glucose were monitored throughout the study. At the end of the study animals were given an intraperitoneal glucose tolerance test. Animals were then culled and blood and tissues were collected for analysis. Insulin and glucagon contents of plasma and tissues were measured by insulin radioimmunoassay and chemiluminescent enzyme-linked immunosorbance assay respectively. Histological analyses of pancreatic islets were carried out by quantitative fluorescence immunohistochemistry staining. RESULTS: Both pseudoislet and cell suspension implants yielded well vascularised ß-cell masses of similar insulin content. This was associated with progressive amelioration of hyperphagia (P < 0.05), polydipsia (P < 0.05), body weight loss (P < 0.05), hypoinsulinaemia (P < 0.05), hyperglycaemia (P < 0.05 - P < 0.001) and glucose tolerance (P < 0.01). Islet morphology was also significantly improved in both groups of transplanted mice, with increased ß-cell (P < 0.05 - P < 0.001) and decreased alpha cell (P < 0.05 - P < 0.001) areas. Whereas mice receiving 1.1B4 cell suspensions eventually exhibited hypoglycaemic complications, pseudoislet recipients displayed a more gradual amelioration of diabetes, and achieved stable blood glucose control similar to non-diabetic mice at the end of the study. CONCLUSION: Although further work is needed to address safety issues, these results provide proof of concept for possible therapeutic applicability of human ß-cell line pseudoislets in diabetes.