Endogenous Β-cell Research Articles

8074 Investigating Viral Infections, Systemic Inflammation, and their Complex Interplay in Type 1 Diabetes Pathogenesis

Abstract Disclosure: K. Girdhar: None. E. Elko: None. A. Pina: None. M. Atkinson: None. J. Ludvigsson: None. J. Ladner: None. E. Altindis: None. Type 1 diabetes (T1D) is an autoimmune disorder characterized by the immune-mediated destruction of insulin-producing beta cells. The etiological factors triggering the immunogenicity of endogenous β-cell antigens remain a focal point of investigation. Viral infections, particularly enteroviruses, coxsackieviruses, and rotaviruses, have been proposed as potential contributors to T1D onset and progression. However, the causal relationship between viral infections and T1D pathogenesis remains elusive. To address this, we conducted an in-depth analysis of viral infection histories of T1D patients and pediatric T1D progressors compared to the controls. To this end, we employed highly multiplexed serology using PepSeq sequencing, an in vitro platform designed for conducting proteomic assays against customizable targets via DNA-barcoded peptides. Utilizing samples from the All Babies in Sweden (ABIS) study (n=58) and the TrialNet study (n=116 new-onset T1D, n=25 established T1D, n=128 controls), we assessed seropositivity against 79 different human viruses. Contrary to previous reports, no significant differences were identified in viral infection histories between T1D individuals and controls. Subsequent analyses of enriched Peptide Count and Viral Homology Search (VHS) species provided further confirmation of the absence of distinctions in T1D progression. Next, we determined whether there are any differences in the markers of systematic inflammation in spite of their similar viral history profile. Therefore, we employed Luminex assays to investigate cytokine profiles in established T1D individuals (n=25). While we could not identify any differences when we compared T1D patients to controls, we identified notable sex-specific differences. Anti-inflammatory cytokines (IL-4, IL-13, IL-22) were lower in female T1D patients compared to female controls. Likewise, female T1D patients had higher pro-inflammatory cytokines (IL-17E, TNF-β). On the other hand, male T1D patients exhibited increased levels of epidermal growth factor and platelet-derived growth factor compared to male controls, with IL-22 uniquely elevated. Our findings challenge the direct association between viral infections and T1D, highlighting the importance of investigating alternative factors. Moreover, the observed sex-specific cytokine alterations underscore the complex interplay between immune responses and T1D, emphasizing the need for sex-specific therapeutic strategies. This research contributes valuable insights into the intricate dynamics of viral infections, systemic inflammation, and their potential roles in T1D pathogenesis. Presentation: 6/2/2024

Mapping of a hybrid insulin peptide in the inflamed islet β-cells from NOD mice.

There is accumulating evidence that pathogenic T cells in T1D recognize epitopes formed by post-translational modifications of β-cell antigens, including hybrid insulin peptides (HIPs). The ligands for several CD4 T-cell clones derived from the NOD mouse are HIPs composed of a fragment of proinsulin joined to peptides from endogenous β-cell granule proteins. The diabetogenic T-cell clone BDC-6.9 reacts to a fragment of C-peptide fused to a cleavage product of pro-islet amyloid polypeptide (6.9HIP). In this study, we used a monoclonal antibody (MAb) to the 6.9HIP to determine when and where HIP antigens are present in NOD islets during disease progression and with which immune cells they associate. Immunogold labeling of the 6.9HIP MAb and organelle-specific markers for electron microscopy were employed to map the subcellular compartment(s) in which the HIP is localized within β-cells. While the insulin B9-23 peptide was present in nearly all islets, the 6.9HIP MAb stained infiltrated islets only in NOD mice at advanced stages of T1D development. Islets co-stained with the 6.9HIP MAb and antibodies to mark insulin, macrophages, and dendritic cells indicate that 6.9HIP co-localizes within insulin-positive β-cells as well as intra-islet antigen-presenting cells (APCs). In electron micrographs, the 6.9HIP co-localized with granule structures containing insulin alone or both insulin and LAMP1 within β-cells. Exposing NOD islets to the endoplasmic reticulum (ER) stress inducer tunicamycin significantly increased levels of 6.9HIP in subcellular fractions containing crinosomes and dense-core granules (DCGs). This work demonstrates that the 6.9HIP can be visualized in the infiltrated islets and suggests that intra-islet APCs may acquire and present HIP antigens within islets.

88-OR: Targeted Drug Delivery to Human ß-Cells Using Novel Nanocapsule Delivery Vehicle for T1D Treatment

In type 1 diabetes (T1D), autoreactive immune cells destroy insulin secreting β-cells in pancreatic islets. Islet transplantation shows promise in restoring glucose homeostasis without exogenous insulin; however, long-term graft survival remains a critical barrier to the success of this treatment. Therapies aimed to protect or proliferate endogenous β-cells have off target affects and require targeted delivery to improve efficacy. We have developed a nanocapsule (NC) delivery system to specifically target and deliver cargo to human β-cells. We hypothesize NCs will selectively be taken up by human β-cells and deliver therapeutic cargo. NCs were synthesized with a water in oil emulsion evaporation technique. NCs were coated via ionic interactions with ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) antibody, a human β-cell specific marker or Exendin-4 conjugated to hyaluronic acid (HA-Ex4). NC size, size distribution, charge, stability, and drug release profiles were determined. NCs loaded with pentamidine (PTM) or Cy5 were cultured for 24-168h with human iPSC-derived β-cells (sBC) or injected into the tail vein of NOD-SCID mice. Confocal microscopy confirmed delivery of cargo to β-cells and pancreatic islets, viability, and cytotoxicity. NCs were 271 ± 7nm in size with excellent size homogeneity (polydispersity index 0.06 ± 0.03, n=3). NCs displayed a sustained release of cargo for >45 days (n=5). In vitro, we observed no cytotoxicity of NCs up to 168h (n=3) and a significant increase in β-cell death with PTM loaded ENTPD3 coated NCs compared to controls at 24 (p<0.001, n=4) and 72h (p<0.01, n=4). sBCs treated with PTM loaded ENTPD3 coated NCs had more insulin+ dead cells (88 ± 4%) than controls (p<0.01, n=4). ENTPD3 or HA-Ex4 NCs were internalized into sBCs in vitro and pancreatic islets in vivo respectively. Our data shows long term stability and in vivo targeting of NCs to β-cells and supports use of our NCs for targeted delivery of therapeutics to human β-cells to treat T1D. Disclosure J.Collins: None. K.Holcomb: None. A.Ocampo: None. A.Shilleh: None. H.A.Russ: Advisory Panel; Sigilon Therapeutics, Inc., Consultant; Prellis, Minutia, Eli Lilly and Company. N.L.Farnsworth: None. Funding JDRF (1-INO-2022-1231-S-B); National Institutes of Health (P30-DK116073)

Pharmacological inhibitors of β-cell dysfunction and death as therapeutics for diabetes.

More than 500 million adults suffer from diabetes worldwide, and this number is constantly increasing. Diabetes causes 5 million deaths per year and huge healthcare costs per year. β-cell death is the major cause of type 1 diabetes. β-cell secretory dysfunction plays a key role in the development of type 2 diabetes. A loss of β-cell mass due to apoptotic death has also been proposed as critical for the pathogenesis of type 2 diabetes. Death of β-cells is caused by multiple factors including pro-inflammatory cytokines, chronic hyperglycemia (glucotoxicity), certain fatty acids at high concentrations (lipotoxicity), reactive oxygen species, endoplasmic reticulum stress, and islet amyloid deposits. Unfortunately, none of the currently available antidiabetic drugs favor the maintenance of endogenous β-cell functional mass, indicating an unmet medical need. Here, we comprehensively review over the last ten years the investigation and identification of molecules of pharmacological interest for protecting β-cells against dysfunction and apoptotic death which could pave the way for the development of innovative therapies for diabetes.

Biomaterials-based nanoparticles conjugated to regulatory T cells provide a modular system for localized delivery of pharmacotherapeutic agents.

Type 1 diabetes (T1D) presents with two therapeutic challenges: the need to correct underlying autoimmunity and restore β-cell mass. We harnessed the unique capacity of regulatory T cells (Tregs) and the T cell receptor (TCR) to direct tolerance induction along with tissue-localized delivery of therapeutic agents to restore endogenous β-cell function. Specifically, we designed a combinatorial therapy involving biomaterials-based poly(lactic-co-glycolic acid) nanoparticles co-loaded with the Treg growth factor, IL-2, and the β-cell regenerative agent, harmine (a tyrosine-regulated kinase 1A [DYRK1A] inhibitor), conjugated to the surface of Tregs. We observed continuous elution of IL-2 and harmine from nanoparticles for at least 7 days in vitro. When conjugated to primary human Tregs, IL-2 nanoparticles provided sufficient IL-2 receptor signaling to support STAT5 phosphorylation for sustained phenotypic stability and viability in culture. Inclusion of poly-L-lysine (PLL) during nanoparticle-cell coupling dramatically increased conjugation efficiency, providing sufficient IL-2 to support in vitro proliferation of IL-2-dependent CTLL-2 cells and primary murine Tregs. In 12-week-old female non-obese diabetic mice, adoptive transfer of IL-2/harmine nanoparticle-conjugated NOD.BDC2.5 Tregs, which express an islet antigen-specific TCR, significantly prevented diabetes demonstrating preserved in vivo viability. These data provide the preclinical basis to develop a biomaterials-optimized cellular therapy to restore immune tolerance and promote β-cell proliferation in T1D through receptor-targeted drug delivery within pancreatic islets.

Comparison of therapeutic effects of mesenchymal stem cells from umbilical cord and bone marrow in the treatment of type 1 diabetes

BackgroundThe therapeutic potential of mesenchymal stem cells (MSCs) in type 1 diabetes (T1D) has been demonstrated in both preclinical and clinical studies. MSCs that have been used in research on T1D are derived from various tissue sources, with bone marrow (BM) and umbilical cord (UC) tissues being the most commonly used. However, the influence of tissue origin on the functional properties and therapeutic effects of MSCs in T1D remains unclear. This study aimed to compare the therapeutic efficacy of UC-MSCs and BM-MSCs in a mouse model of T1D as well as in patients with T1D.MethodsIn non-obese diabetic (NOD) mice, the development of diabetes was accelerated by streptozotocin injections. Thereafter, diabetic mice were randomized and treated intravenously with UC-MSCs, BM-MSCs or phosphate-buffered saline as a control. Blood glucose and serum insulin were measured longitudinally after transplantation. At 14 days post-transplantation, pancreatic tissues were collected to assess insulitis and the β-cell mass. Flow cytometry was performed to evaluate the composition of T lymphocytes in the spleen and pancreatic lymph nodes of the NOD mice. In our retrospective study of patients with T1D, 28 recipients who received insulin therapy alone or a single transplantation of UC-MSCs or BM-MSCs were enrolled. The glycaemic control and β-cell function of the patients during the first year of follow-up were compared.ResultsIn NOD mice, UC-MSC and BM-MSC transplantation showed similar effects on decreasing blood glucose levels and preserving β cells. The regulation of islet autoimmunity was examined, and no significant difference between UC-MSCs and BM-MSCs was observed in the attenuation of insulitis, the decrease in T helper 17 cells or the increase in regulatory T cells. In patients with T1D, MSC transplantation markedly lowered haemoglobin A1c (HbA1c) levels and reduced insulin doses compared to conventional insulin therapy. However, the therapeutic effects were comparable between UC-MSCs and BM-MSCs, and they also exerted similar effects on the endogenous β-cell function in the patients.ConclusionIn conclusion, both UC-MSCs and BM-MSCs exhibited comparable therapeutic effects on improving glycaemic control and preserving β-cell function in T1D. Considering their abundance and higher cell yields, UC-MSCs appear to be more promising than BM-MSCs in clinical applications.Trial registration NCT02763423. Registered on May 5, 2016—Retrospectively registered, https://www.clinicaltrials.gov/.

Verapamil and Its Role in Diabetes

Autoimmune pancreatic β-cell loss and destruction play a key role in the pathogenesis and development of type 1 diabetes, with a prospective increased risk for developing micro- and macrovascular complications. In this regard, orally administrated verapamil, a calcium channel antagonist, usually intended for use as an anti-arrhythmic drug, has previously shown potential beneficial effects on β-cell preservation in new-onset type 1 diabetes. Furthermore, observational data suggest a reduced risk of type 2 diabetes development. The underlying pathophysiological mechanisms are not well investigated and remain widely inconclusive. The aim of this narrative review was to detail the role of verapamil in promoting endogenous β-cell function, potentially eligible for early treatment in type 1 diabetes, and to summarize existing evidence on its effect on glycemia in individuals with type 2 diabetes.

Gymnemic Acid Ameliorates Pancreatic β-Cell Dysfunction by Modulating Pdx1 Expression: A Possible Strategy for β-Cell Regeneration.

Endogenous pancreatic β-cell regeneration is a promising therapeutic approach for enhancing β-cell function and neogenesis in diabetes. Various findings have reported that regeneration might occur via stimulating β-cell proliferation, neogenesis, or conversion from other pancreatic cells to β-like cells. Although the current scenario illustrates numerous therapeutic strategies and approaches that concern endogenous β-cell regeneration, all of them have not been successful to a greater extent because of cost effectiveness, availability of suitable donors and rejection in case of transplantation, or lack of scientific evidence for many phytochemicals derived from plants that have been employed in traditional medicine. Therefore, the present study aims to investigate the effect of gymnemic acid (GA) on β-cell regeneration in streptozotocin-induced type 1 diabetic rats and high glucose exposed RIN5-F cells. The study involves histopathological and immunohistochemical analysis to examine the islet's architecture. Quantitative polymerase chain reaction (qPCR) and/or immunoblot were employed to quantify the β-cell regeneration markers and cell cycle proliferative markers. The immunoexpression of E-cadherin, β-catenin, and phosphoinositide 3-kinases/protein kinase B were significantly increased in GA-treated diabetic rats. On the other hand, treatment with GA upregulated the pancreatic regenerative transcription factor viz. pancreatic duodenal homeobox 1, Neurogenin 3, MafA, NeuroD1, and β-cells proliferative markers such as CDK4, and Cyclin D1, with a simultaneous downregulation of the forkhead box O, glycogen synthase kinase-3, and p21cip1 in diabetic treated rats. Adding to this, we noticed increased nuclear localization of Pdx1 in GA treated high glucose exposed RIN5-F cells. Our results suggested that GA acts as a potential therapeutic candidate for endogenous β-cell regeneration in treating type 1 diabetes.

Direct Reprogramming of Somatic Cells into Induced β-Cells: An Overview.

The persistent shortage of insulin-producing islet mass or β-cells for transplantation in the ever-growing diabetic population worldwide is a matter of concern. To date, permanent cure to this medical complication is not available and soon after the establishment of lineage-specific reprogramming, direct β-cell reprogramming became a viable alternative for β-cell regeneration. Direct reprogramming is a straightforward and powerful technique that can provide an unlimited supply of cells by transdifferentiating terminally differentiated cells toward the desired cell type. This approach has been extensively used by multiple groups to reprogram non-β-cells toward insulin-producing β-cells. The β-cell identity has been achieved by various studies via ectopic expression of one or more pancreatic-specific transcription factors in somatic cells, bypassing the pluripotent state. This work highlights the importance of the direct reprogramming approaches (both integrative and non-integrative) in generating autologous β-cells for various applications. An in-depth understanding of the strategies and cell sources could prove beneficial for the efficient generation of integration-free functional insulin-producing β-cells for diabetic patients lacking endogenous β-cells.

Peptide-YY3-36/glucagon-like peptide-1 combination treatment of obese diabetic mice improves insulin sensitivity associated with recovered pancreatic β-cell function and synergistic activation of discrete hypothalamic and brainstem neuronal circuitries

ObjectiveObesity-linked type 2 diabetes (T2D) is a worldwide health concern and many novel approaches are being considered for its treatment and subsequent prevention of serious comorbidities. Co-administration of glucagon like peptide 1 (GLP-1) and peptide YY3-36 (PYY3-36) renders a synergistic decrease in energy intake in obese men. However, mechanistic details of the synergy between these peptide agonists and their effects on metabolic homeostasis remain relatively scarce. MethodsIn this study, we utilized long-acting analogues of GLP-1 and PYY3-36 (via Fc-peptide conjugation) to better characterize the synergistic pharmacological benefits of their co-administration on body weight and glycaemic regulation in obese and diabetic mouse models. Hyperinsulinemic-euglycemic clamps were used to measure weight-independent effects of Fc-PYY3-36 + Fc-GLP-1 on insulin action. Fluorescent light sheet microscopy analysis of whole brain was performed to assess activation of brain regions. ResultsCo-administration of long-acting Fc-IgG/peptide conjugates of Fc-GLP-1 and Fc-PYY3-36 (specific for PYY receptor-2 (Y2R)) resulted in profound weight loss, restored glucose homeostasis, and recovered endogenous β-cell function in two mouse models of obese T2D. Hyperinsulinemic-euglycemic clamps in C57BLKS/J db/db and diet-induced obese Y2R-deficient (Y2RKO) mice indicated Y2R is required for a weight-independent improvement in peripheral insulin sensitivity and enhanced hepatic glycogenesis. Brain cFos staining demonstrated distinct temporal activation of regions of the hypothalamus and hindbrain following Fc-PYY3-36 + Fc-GLP-1R agonist administration. ConclusionsThese results reveal a therapeutic approach for obesity/T2D that improved insulin sensitivity and restored endogenous β-cell function. These data also highlight the potential association between the gut–brain axis in control of metabolic homeostasis.

Engineering islets from stem cells for advanced therapies of diabetes.

Diabetes mellitus is a metabolic disorder that affects more than 460 million people worldwide. Type 1 diabetes (T1D) is caused by autoimmune destruction of β-cells, whereas type 2 diabetes (T2D) is caused by a hostile metabolic environment that leads to β-cell exhaustion and dysfunction. Currently, first-line medications treat the symptomatic insulin resistance and hyperglycaemia, but do not prevent the progressive decline of β-cell mass and function. Thus, advanced therapies need to be developed that either protect or regenerate endogenous β-cell mass early in disease progression or replace lost β-cells with stem cell-derived β-like cells or engineered islet-like clusters. In this Review, we discuss the state of the art of stem cell differentiation and islet engineering, reflect on current and future challenges in the area and highlight the potential for cell replacement therapies, disease modelling and drug development using these cells. These efforts in stem cell and regenerative medicine will lay the foundations for future biomedical breakthroughs and potentially curative treatments for diabetes.

Genetic Composition and Autoantibody Titers Model the Probability of Detecting C-Peptide Following Type 1 Diabetes Diagnosis.

We and others previously demonstrated that a type 1 diabetes genetic risk score (GRS) improves the ability to predict disease progression and onset in at-risk subjects with islet autoantibodies. Here, we hypothesized that GRS and islet autoantibodies, combined with age at onset and disease duration, could serve as markers of residual β-cell function following type 1 diabetes diagnosis. Generalized estimating equations were used to investigate whether GRS along with insulinoma-associated protein-2 autoantibody (IA–2A), zinc transporter 8 autoantibody (ZnT8A), and GAD autoantibody (GADA) titers were predictive of C-peptide detection in a largely cross-sectional cohort of 401 subjects with type 1 diabetes (median duration 4.5 years [range 0–60]). Indeed, a combined model with incorporation of disease duration, age at onset, GRS, and titers of IA–2A, ZnT8A, and GADA provided superior capacity to predict C-peptide detection (quasi-likelihood information criterion [QIC] = 334.6) compared with the capacity of disease duration, age at onset, and GRS as the sole parameters (QIC = 359.2). These findings support the need for longitudinal validation of our combinatorial model. The ability to project the rate and extent of decline in residual C-peptide production for individuals with type 1 diabetes could critically inform enrollment and benchmarking for clinical trials where investigators are seeking to preserve or restore endogenous β-cell function.

Proinsulin-specific T-cell responses correlate with estimated c-peptide and predict partial remission duration in type 1 diabetes.

ObjectiveType 1 diabetes (T1D) is an autoimmune disorder in which autoreactive T cells destroy insulin‐producing β‐cells. Interventions that preserve β‐cell function represent a fundamental therapeutic goal in T1D and biomarkers that predict and monitor β‐cell function, and changes in islet autoantigenic signatures are needed. As proinsulin and neoantigens derived from proinsulin peptides (hybrid insulin peptides, HIPs) are important T1D autoantigens, we analysed peripheral blood CD4+ T‐cell autoantigen‐specific proliferative responses and their relationship to estimated β‐cell function.MethodsWe recruited 72 people with and 42 without T1D, including 17 pre‐diabetic islet antibody‐positive and 9 antibody‐negative first‐degree relatives and 16 unrelated healthy controls with T1D‐risk HLA types. We estimated C‐peptide level at 3‐month intervals for 2 years post‐diagnosis and measured CD4+ T‐cell proliferation to proinsulin epitopes and HIPs using an optimised bioassay.ResultsWe show that CD4+ T‐cell proliferation to any islet peptide and to multiple epitopes were significantly more frequent in pre‐diabetic islet antibody‐positive siblings and participants with T1D ≤ 3 months of duration, than in participants with T1D > 3 months or healthy controls. Among participants with T1D and first‐degree relatives, CD4+ T‐cell proliferation occurred most frequently in response to proinsulin33‐63 (full‐length C‐peptide). Proinsulin33‐63‐specific responses were associated with HLA‐DR3‐DQ2 and/or HLA‐DR4/DQ8. In children with T1D, proinsulin33‐63‐specific T‐cell proliferation positively associated with concurrent estimated C‐peptide and predicted survival in honeymoon.ConclusionCD4+ T‐cell proliferative responses to proinsulin‐containing autoantigens are common before and immediately after diagnosis of T1D but decline thereafter. Proinsulin33‐63‐specific CD4+ T‐cell response is a novel marker of estimated residual endogenous β‐cell function and predicts a better 2‐year disease outcome.

InsB9-23 Gene Transfer to Hepatocyte-Based Combined Therapy Abrogates Recurrence of Type 1 Diabetes After Islet Transplantation.

The induction of antigen (Ag)-specific tolerance represents a therapeutic option for autoimmune diabetes. We demonstrated that administration of a lentiviral vector enabling expression of insulin B chain 9-23 (InsB9-23) (LV.InsB) in hepatocytes arrests β-cell destruction in prediabetic NOD mice by generating InsB9-23-specific FoxP3+ T regulatory cells (Tregs). LV.InsB in combination with a suboptimal dose of anti-CD3 monoclonal antibody (combined therapy [CT], 1 × 5 μg [CT5]) reverts diabetes and prevents recurrence of autoimmunity after islet transplantation in ∼50% of NOD mice. We investigated whether CT optimization could lead to abrogation of recurrence of autoimmunity. Therefore, alloislets were transplanted after optimized CT tolerogenic conditioning (1 × 25 μg [CT25]). Diabetic NOD mice conditioned with CT25 when glycemia was <500 mg/dL remained normoglycemic for 100 days after alloislet transplantation and displayed reduced insulitis, but independently from the graft. Accordingly, cured mice showed T-cell unresponsiveness to InsB9-23 stimulation and increased Treg frequency in islet infiltration and pancreatic lymph nodes. Additional studies revealed a complex mechanism of Ag-specific immune regulation driven by CT25, in which both Tregs and PDL1 costimulation cooperate to control diabetogenic cells, while transplanted islets play a crucial role, although transient, recruiting diabetogenic cells. Therefore, CT25 before alloislet transplantation represents an Ag-specific immunotherapy to resolve autoimmune diabetes in the presence of residual endogenous β-cell mass.

Staprexanthones, Xanthone-Type Stimulators of Pancreatic β-Cell Proliferation from a Mangrove Endophytic Fungus.

This project was focused on the discovery of novel compounds that promote endogenous β-cell regeneration. Screening of extracts identified the fungus Stachybotrys chartarum as a promising candidate. After fermentation and extraction of S. chartarum, we isolated five new prenylated xanthones, namely, staprexanthones A-E (1-5), with staprexanthone A (1) being the first natural xanthone bearing a rare 4,5-dimethyl-1,3-dioxolane moiety. Compounds 1, 2, and 5 significantly increased β-cell numbers in vivo in a zebrafish model. Further analysis revealed that 2 and 5 promoted β-cell mass expansion by increasing proliferation of existing β-cells though promotion of cell-cycle progression at the G1/S transition. These findings indicate that prenylated xanthones are potential new drug leads for antidiabetes therapy by stimulating β-cell regeneration.

1800-P: Cotadutide, a GLP-1/GCG Receptor Co-Agonist, Improves Insulin Sensitivity and Restores Normal Insulin Secretory Capacity in DIO Mice

Cotadutide (MEDI0382) is a peptide with targeted GLP-1/glucagon (Gcg) receptor activity that has beneficial effects on T2D and obesity in humans and mice, and shows marked improvements in NAFLD/NASH in mice. We performed hyperinsulinemic clamps in diet induced obese (DIO) mice to better understand whether improved insulin sensitivity and β-cell function are responsible for the improved glycemia in our clinical studies. Following 28 days of daily dosing with Cotadutide (10 nmol/kg), Liraglutide (5 nmol/kg; GLP-1 agonist) or g1437 (5 nmol/kg; Gcg analog) mice underwent continuous infusion of 4mU/kg/min insulin and [3H]-glucose to assess glucose turnover. Tissue-specific glucose uptake was assessed using 14C-2-deoxyglucose. We observed lower body weight in all groups compared with vehicle control, with g1437 eliciting the largest response and Liraglutide the least. Fasting glucose was significantly higher in g1437 treated mice. Fasting insulin was dramatically lower in Cotadutide and g1437 groups compared with vehicle. Despite an equal rate of human insulin infusion between groups, the total insulin levels during the clamp remained lower in Cotadutide and g1437 treated mice. The glucose infusion rate, to maintain euglycemia ∼130 mg/dL, was significantly higher in Cotadutide treated mice in line with increased glucose disposal rate. Hepatic glucose production was suppressed in all groups, despite significantly lower levels of insulin in Cotadutide and g1437 treated mice. Finally, glucose uptake was elevated in brown adipose tissue in Cotadutide treated mice, which was confirmed in a separate study of [18F]FDG uptake by PET imaging. These experiments demonstrate that Cotadutide improved insulin sensitivity in concert with dramatic reductions for insulin demand, which may result in recovery of endogenous β-cell function in T2D. Disclosure R.C. Laker: Employee; Self; AstraZeneca. L. Lantier: None. O. McGuinness: None. S. Will: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. K. Kuszpit: None. A.R. Alfaro: None. L. Jermutus: Employee; Self; AstraZeneca. Stock/Shareholder; Self; AstraZeneca. C.J. Rhodes: Employee; Self; AstraZeneca.

1774-P: Neurally Mediated Prandial Islet-Cell Function Is Glucose-Independent and Preserved after Gastric Bypass and Sleeve Gastrectomy

The weight-loss independent glycemic effects of gastric bypass (GB) are characterized by prandial short-lived hyperinsulinemia as a result of β-cell stimulation by glucose and incretins. To investigate the effect of parasympathetic nervous system (PNS) activity, a neural component of the enteroinsular axis, on islet function, 9 subjects with history of GB, 6 with prior sleeve gastrectomy (SG), and 4 non-surgical controls were recruited. The GB, SG, and CN groups were matched for BMI, FFM, A1C, and age; and the GB and SG for weight loss and time postsurgery; none had diabetes. To measure islet hormone responses to meal ingestion when the effect of GI hormones and glycemia is minimal, subjects consumed a liquid meal (280 kcal, 20% glucose) at 2 hr of a 5-hr hyperinsulinemic (120 mU/m2/min) hypoglycemic (55mg/dl) clamp with and without atropine infusion on two separate studies. Fasting levels of glucose, islet and gut hormones were similar among 3 groups and 2 studies. Despite similar insulin sensitivity among the groups the insulin clearance estimated before meal ingestion was larger in surgical patients (P=0.08). Suppression of endogenous β-cell secretion by hypoglycemia was less in both surgical groups compared to controls (P&amp;lt;0.001), but it was reduced by atropine in 3 groups (P&amp;lt;0.05). Hypoglycemia-induced glucagon secretion was not affected by PNS blockade. Blocking PNS diminished both β- and α-cell secretory response to meal ingestion (P &amp;lt; 0.05) in both surgical and non-surgical groups. Pre- and post-meal pancreatic polypeptide, marker of vagal input to the islet, declined to baseline values during atropine infusion. Meal-induced GIP secretion was similar in studies with and without atropine whereas blocking PNS significantly reduced GLP-1 secretion (P &amp;lt; 0.05) in all subjects. Our data shows that in nondiabetic subjects with prior GB and SG, PNS activity regulates islet and gut hormone secretory response to meal ingestion independent of glucose levels. Disclosure H. Honka: None. A.M. Al Zubaidi: None. R.A. DeFronzo: None. A. Gastaldelli: Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Gilead Sciences, Inc., Inventiva Pharma, Novo Nordisk Inc. M. Salehi: None. Funding Finnish Cultural Foundation (00180071); National Institutes of Health (DK105379)

Characterization of Signaling Pathways Associated with Pancreatic β-cell Adaptive Flexibility in Compensation of Obesity-linked Diabetes in db/db Mice

The onset of obesity-linked type 2 diabetes (T2D) is marked by an eventual failure in pancreatic β-cell function and mass that is no longer able to compensate for the inherent insulin resistance and increased metabolic load intrinsic to obesity. However, in a commonly used model of T2D, the db/db mouse, β-cells have an inbuilt adaptive flexibility enabling them to effectively adjust insulin production rates relative to the metabolic demand. Pancreatic β-cells from these animals have markedly reduced intracellular insulin stores, yet high rates of (pro)insulin secretion, together with a substantial increase in proinsulin biosynthesis highlighted by expanded rough endoplasmic reticulum and Golgi apparatus. However, when the metabolic overload and/or hyperglycemia is normalized, β-cells from db/db mice quickly restore their insulin stores and normalize secretory function. This demonstrates the β-cell's adaptive flexibility and indicates that therapeutic approaches applied to encourage β-cell rest are capable of restoring endogenous β-cell function. However, mechanisms that regulate β-cell adaptive flexibility are essentially unknown. To gain deeper mechanistic insight into the molecular events underlying β-cell adaptive flexibility in db/db β-cells, we conducted a combined proteomic and post-translational modification specific proteomic (PTMomics) approach on islets from db/db mice and wild-type controls (WT) with or without prior exposure to normal glucose levels. We identified differential modifications of proteins involved in redox homeostasis, protein refolding, K48-linked deubiquitination, mRNA/protein export, focal adhesion, ERK1/2 signaling, and renin-angiotensin-aldosterone signaling, as well as sialyltransferase activity, associated with β-cell adaptive flexibility. These proteins are all related to proinsulin biosynthesis and processing, maturation of insulin secretory granules, and vesicular trafficking-core pathways involved in the adaptation of insulin production to meet metabolic demand. Collectively, this study outlines a novel and comprehensive global PTMome signaling map that highlights important molecular mechanisms related to the adaptive flexibility of β-cell function, providing improved insight into disease pathogenesis of T2D.

Transplantation of human dental pulp stem cells in streptozotocin-induced diabetic rats.

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease caused by the destruction of pancreatic β-cells. Human dental pulp stem cells represent a promising source for cell-based therapies, owing to their easy, minimally invasive surgical access, and high proliferative capacity. It was reported that human dental pulp stem cells can differentiate into a pancreatic cell lineage in vitro; however, few studies have investigated their effects on diabetes. Our study aimed to investigate the therapeutic potential of intravenous and intrapancreatic transplantation of human dental pulp stem cells in a rat model of streptozotocin-induced type 1 diabetes. Forty Sprague Dawley male rats were randomly categorized into four groups: control, diabetic (STZ), intravenous treatment group (IV), and intrapancreatic treatment group (IP). Human dental pulp stem cells (1 × 106cells) or vehicle were injected into the pancreas or tail vein 7days after streptozotocin injection. Fasting blood glucose levels were monitored weekly. Glucose tolerance test, rat and human serum insulin and C-peptide, pancreas histology, and caspase-3, vascular endothelial growth factor, and Ki67 expression in pancreatic tissues were assessed 28days post-transplantation. We found that both IV and IP transplantation of human dental pulp stem cells reduced blood glucose and increased levels of rat and human serum insulin and C-peptide. The cells engrafted and survived in the streptozotocin-injured pancreas. Islet-like clusters and scattered human dental pulp stem cells expressing insulin were observed in the pancreas of diabetic rats with some difference in the distribution pattern between the two injection routes. RT-PCR analyses revealed the expression of the human-specific pancreatic β-cell genes neurogenin 3 (NGN3), paired box 4 (PAX4), glucose transporter2 (GLUT2), and insulin in the pancreatic tissues of both the IP and IV groups. In addition, the transplanted cells downregulated the expression of caspase-3 and upregulated the expression of vascular endothelial growth factor and Ki67, suggesting that the injected cells exerted pro-angiogenetic and antiapoptotic effects, and promoted endogenous β-cell replication. Our study is the first to show that human dental pulp stem cells can migrate and survive within streptozotocin-injured pancreas, and induce antidiabetic effects through the differentiation and replacement of lost β-cells and paracrine-mediated pancreatic regeneration. Thus, human dental pulp stem cells may have therapeutic potential to treat patients with long term T1DM.

Interleukin-2 Therapy of Autoimmunity in Diabetes (ITAD): a phase 2, multicentre, double-blind, randomized, placebo-controlled trial

Type 1 diabetes is a common autoimmune disease due to destruction of pancreatic β cells, resulting in lifelong need for insulin. Evidence suggest that maintaining residual β-cell function can improve glucose control and reduce risk of hypoglycaemia and vascular complications. Non-clinical, preclinical and some preliminary clinical data suggest that low-dose interleukin-2 (IL-2) therapy could block pancreatic β cells destruction by increasing the number of functional regulatory T cells (Tregs) that inhibit islet-specific autoreactive effector T cells (Teffs). However, there is lack of data on the effect of low-dose IL-2 in newly diagnosed children and adolescents with T1D as well as lack of specific data on its potential effect on β-cell function. The ‘ Interleukin-2 Therapy of Autoimmunity in Diabetes (ITAD)’ is a phase 2, multicentre, double-blind, randomised, placebo-controlled trial in children and adolescents (6-18 years; having detectable C-peptide) initiated within 6 weeks of T1D diagnosis. A total of 45 participants will be randomised in a 2:1 ratio to receive either ultra-low dose IL-2 (aldesleukin), at a dose of 0.2 x 10 6 IU/m 2 twice-weekly, given subcutaneously, or placebo, for 6 months. The primary objective is to assess the effects of ultra-low dose aldesleukin administration on endogenous β-cell function as measured by frequent home dried blood spot (DBS) fasting and post-prandial C-peptide in children and adolescents with newly diagnosed T1D. The secondary objectives are: 1) to assess the efficacy of regular dosing of aldesleukin in increasing Treg levels; 2) to confirm the clinical safety and tolerability of ultra-low dose aldesleukin; 3) to assess changes in the immune system indicating benefit or potential risk for future gains/loss in β-cell function and immune function; 4) to assess treatment effect on glycaemic control. Trial registration: EudraCT 2017-002126-20 (06/02/2019)