Islet Mass Research Articles

1686-P: Brown Adipose Tissue as a Novel Site for Islet Transplantation

Type 1 diabetes (T1D) results in hyperglycemia, increased risk for secondary complications, and life-long dependence on exogenous insulin. Islet transplantation is a promising alternative to insulin therapy for T1D treatment. However, long-term function of islets transplanted into the FDA approved hepatic portal vein remains low, with much of the islet mass being lost early after transplantation due to immune responses and drug toxicity. These challenges have prompted a search for alternative transplantation sites that are readily accessible, immune-privileged, offer graft retrievability, and promote islet survival. A novel site that exhibits these traits is brown adipose tissue (BAT), which is highly vascularized, innervated, and contains immunomodulatory immune cells. BAT functions to maintain thermogenesis and can regulate metabolic homeostasis. We hypothesize that islet engraftment in BAT can restore euglycemia in diabetic mice, promote revascularization, and delay immune rejection. Syngeneic transplantation of 250 NOD.scid islets into the BAT of streptozotocin (STZ)-treated diabetic NOD.scid mice restored euglycemia for over two months, similar to islets transplanted under the pre-clinical kidney capsule transplant site. Islet function was also comparable across transplant groups as measured by a glucose tolerance test. BAT thermogenic function during a cold challenge showed no difference in cold tolerance after transplantation. Histology and mRNA analysis revealed BAT engrafted islets expressed islet hormones, functional islet transcription factors, and CD31+ vascularization at the islet graft site. Overall, our data suggest BAT is a novel and efficacious site for islet transplantation. Future islet transplant studies will determine if the inherent properties of BAT can extend the longevity of transplanted islets by investigating the immunomodulatory capacity of BAT-resident mesenchymal stem cells and immune cells, compared to current transplantation sites. Disclosure J. Barra: None. J. Kepple: None. H.M. Tse: None. C.S. Hunter: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK099550); JDRF (SRA-2016-270-S-B, 2-SRA-2019-692-S-B); National Institute of General Medical Sciences (T32GM008111)

The role of total pancreatectomy with islet autotransplantation in the treatment of chronic pancreatitis: A report from the International Consensus Guidelines in chronic pancreatitis

BackgroundAdvances in our understanding of total pancreatectomy with islet autotransplantation (TPIAT) have been made. We aimed to define indications and outcomes of TPIAT. MethodsExpert physician-scientists from North America, Asia, and Europe reviewed the literature to address six questions selected by the writing group as high priority topics. A consensus was reached by voting on statements generated from the review. ResultsConsensus statements were voted upon with strong agreement reached that (Q1) TPIAT may improve quality of life, reduce pain and opioid use, and potentially reduce medical utilization; that (Q3) TPIAT offers glycemic benefit over TP alone; that (Q4) the main indication for TPIAT is disabling pain, in the absence of certain medical and psychological contraindications; and that (Q6) islet mass transplanted and other disease features may impact diabetes mellitus outcomes. Conditional agreement was reached that (Q2) the role of TPIAT for all forms of CP is not yet identified and that head-to-head comparative studies are lacking, and that (Q5) early surgery is likely to improve outcomes as compared to late surgery. ConclusionsAgreement on TPIAT indications and outcomes has been reached through this working group. Further studies are needed to answer the long-term outcomes and maximize efforts to optimize patient selection.

Trisected pancreas model for testing tissue dissociation enzyme combinations: a novel methodology for improving human islet yield for clinical islet transplantation.

Human islet isolation requires a defined collagenase-protease enzyme combination for obtaining a successful islet yield. While different islet laboratories use different enzyme combinations, a systematic methodology to identify optimal enzyme combinations and their concentrations within a single donor pancreas has not been tested. In this study, we designed a trisected pancreas model to test efficacy of three clinical grade enzyme blends (VitaCyte, Roche, SERVA) within a single pancreas. Islet isolations were performed using brain-dead donor pancreases (n = 15) applying the enzyme-related design of experiments (DOEs) and the trisected model approach. After trimming, split each pancreas into three individual lobes (head, body, tail). As per the DOEs, the lobes were altered between different experiments, to minimize anatomical bias. Islets isolated from each lobe (27 lobes totally) were subjected to functional assessments. Insulin staining and islet area fraction were determined for tissue sections obtained from each lobe. Utilizing the trisected model, we identified that the collagenase dose from three different vendors did not affect the pancreas digestion and islet yield, but islet morphology after isolation with the neutral protease and BP-protease was better than thermolysin. In addition, the head lobe yielded a lower islet mass and higher tissue volume compared to other two lobes, irrespective of enzyme combination used. This study demonstrates that the trisected model is a promising methodology in assessing donor and isolation associated parameters. Based on this study, we conclude that the donor characteristics and an optimal enzyme dose play a critical role in achieving higher islet yields.

Pancreatic Islets Aging in Old Rhesus Monkey

BackgroundType 2 diabetes is an age associated disease which characterized by insulin resistance and loss of β cell mass and function. Accumulation of senescent β cells with age probably lead to compromised capacity of insulin synthesis and secretion. Nevertheless, other studies also suggested systemic aging may impair glucose tolerance via compromised peripheral insulin sensitivity and microcirculation. So, the pathophysiological process of pancreatic β cell senescence, and the relationship between senescent β cells and diabetes are not clarified. Rhesus monkey is one of the most ideal non‐human primate models of human aging. To date, the researches on islets function of rhesus monkeys, particularly aged monkeys are so limited. So, in this study, we are aiming to explore the link between islets senescence and diabetes in aged rhesus monkeys of approximately 20 years old that may reflect the characters of humans around 80 years old.MethodsSix middle age rhesus monkeys (average 10 years old) and six old rhesus monkeys (average 20 years old) were used in this study. Blood chemistry was detected and the intravenous glucose tolerance tests (IVGTT) were carried out. After sacrifice, the pancreatic islets were isolated to evaluate β cell function, and the histopathological analysis was done on the pancreatic tissues.ResultsIn the blood chemistry, no significant differences in kidney and liver functions were observed between the two groups. The old monkeys show slight increase on the fasting blood glucose, while their fasting insulin levels were increased distinctly. In addition, the IVGTT results showed that the glucose tolerance of the old group was slightly impaired, indicating the possible occurrence of peripheral insulin resistance. In respect to pancreatic histology, the immunohistochemistry staining with anti‐insulin antibody showed that both the total area of insulin producing β cells in pancreatic sections and the single islet mass decreased a lot in old rhesus monkeys, but interestingly, a notable elevated expression of pancreatic/duodenal homeobox‐1(PDX1), an important biomarker for β cell function, was observed. In respect to isolated islets, no significant difference was found in either glucose‐stimulated insulin secretion test(GSIS) or β‐Gal staining, suggesting the islets may function well in these old monkeys.ConclusionIn our 20 years old rhesus monkeys, the insulin resistance has appeared, and their islets area decreased obviously but not accompany significantly compromise of islets function, suggesting that the islets of old rhesus monkey may still have a strong compensatory ability.Support or Funding InformationThis study was supported by the Program of National Natural Science Foundation of China (81870609, 31571474, and 81571808), and the National Key Clinical Project.A, B, C) The fasting blood glucose, fasting blood insulin and intravenous glucose tolerance tests of middle age and old rhesus monkey. D) The glucose‐stimulated insulin secretion test (GSIS) of isolated isletsFigure 1E) The pancreatic sections immunohistochemistry staining with anti‐insulin antibody of middle age(left) and old(right) rhesus monkeyFigure 2

Improving Clinical Islet Transplantation Outcomes.

Transplantation of islets purified from donor pancreata is one of only a few clinical strategies that can reverse type 1 diabetes (T1D) and restore glycemic control (1–3). Significant progress has been made by improving transplantation strategies in areas including immune suppressive regimes, islet quality, transplanted β-cell mass and purity, and donor selection (4–6). However, while improved metabolic control and reduced glycemic side effects such as hypoglycemia persist long term in the majority of T1D patients, insulin independence beyond 1 year is only achieved in a minority of cases (2). Early loss of islet mass upon portal vein infusion is believed to have a major negative impact (7). Recurrence of insulitis, the hallmark of T1D immunopathogenesis leading to autoimmune mediated β-cell destruction, and islet allograft rejection pose additional therapeutic hurdles (8,9). Recently, neutrophils were suggested as part of the innate immune system contributing to development of T1D (10). Chemokines produced by distressed islet tissue attract leukocytes to the site of implantation (Fig. 1). The chemokine CXCL8, produced by recruited and resident macrophages, attracts neutrophils carrying its receptors CXCR1/2 to the lesion. In this issue of Diabetes Care , Piemonti and colleagues (11) now report the results of a phase 3 clinical trial assessing whether interference in homing of leukocytes to the implanted islet graft using reparixin, a small molecule that interferes with CXCR1/2 signaling, may improve transplantation outcome. The rationale for this strategy is outstanding. Early studies in mice and humans were very encouraging. Treatment with reparixin prevented and reversed autoimmune diabetes in NOD mice and improved graft survival …

Notch1 Has an Important Role in β-Cell Mass Determination and Development of Diabetes

BackgroundNotch signaling pathway plays an important role in regulating pancreatic endocrine and exocrine cell fate during pancreas development. Notch signaling is also expressed in adult pancreas. There are few studies on the effect of Notch on adult pancreas. Here, we investigated the role of Notch in islet mass and glucose homeostasis in adult pancreas using Notch1 antisense transgenic (NAS).MethodsWestern blot analysis was performed for the liver of 8-week-old male NAS mice. We also conducted an intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test in 8-week-old male NAS mice and male C57BL/6 mice (control). Morphologic observation of pancreatic islet and β-cell was conducted in two groups. Insulin secretion capacity in islets was measured by glucose-stimulated insulin secretion (GSIS) and perifusion.ResultsNAS mice showed higher glucose levels and lower insulin secretion in IPGTT than the control mice. There was no significant difference in insulin resistance. Total islet and β-cell masses were decreased in NAS mice. The number of large islets (≥250 µm) decreased while that of small islets (<250 µm) increased. Reduced insulin secretion was observed in GSIS and perifusion. Neurogenin3, neurogenic differentiation, and MAF bZIP transcription factor A levels increased in NAS mice.ConclusionOur study provides that Notch1 inhibition decreased insulin secretion and decreased islet and β-cell masses. It is thought that Notch1 inhibition suppresses islet proliferation and induces differentiation of small islets. In conclusion, Notch signaling pathway may play an important role in β-cell mass determination and diabetes.

Local release of NECA (5′-(N-ethylcarboxamido)adenosine) from implantable polymeric sheets for enhanced islet revascularization in extrahepatic transplantation site

Clinical intraportal pancreatic islet infusion is popular for treating type I diabetes. However, multiple doses of islets and anti-rejection protocols are needed to compensate for early large cell losses post-infusion due to the harsh hepatic environment. Thus, extrahepatic sites are utilized to enable efficient islet engraftment and reduce islet mass. Here, we reported an effective islet revascularization protocol that was based on the co-implantation of islet/fibrin gel construct with poly(lactic-co-glycolic) acid sheet releasing NECA (5′-(N-ethylcarboxamido) adenosine; a potent agonist of adenosine) into mouse epididymal fat pad. Thin, flexible sheets (d = 4 mm) prepared by simple casting exhibited sustained NECA release for up to 21 days, which effectively improved early islet engraftment with a median diabetic reversal time of 18.5 days. Western blotting revealed the facilitative effect of NECA on VEGF expression from islets in vitro and from grafts in vivo. In addition, NECA directly promoted the angiogenic activities of islet-derived endothelial cells by enhancing their proliferation and vessel-like tube formation. As a result, neovasculatures were effectively formed in the engrafted islet vicinity, as evidenced by vasculature imaging and immunofluorescence. Taken together, we suggest NECA-releasing PLGA sheets offer a safe and effective drug delivery system that enhances islet engraftment while reducing islet mass at extrahepatic sites for clinical relevance.

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets.

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Beta-cell β1 integrin deficiency affects in utero development of islet growth and vascularization.

The β1 integrin subunit contributes to pancreatic beta cell growth and function through communication with the extracellular matrix (ECM). The effects of in vitro and in vivo β1 integrin knockout have been extensively studied in mature islets, yet no study to date has examined how the loss of β1 integrin during specific stages of pancreatic development impacts beta cell maturation. Beta-cell-specific tamoxifen-inducible Cre recombinase (MIP-CreERT) mice were crossed with mice containing floxed Itgb1 (β1 integrin) to create an inducible mouse model (MIPβ1KO) at the second transition stage (e13.5)of pancreas development. By e19.5-20.5, the expression of beta-cell β1 integrin in fetal MIPβ1KO mice was significantly reduced and these mice displayed decreased beta cell mass, density and proliferation. Morphologically, fetal MIPβ1KO pancreata exhibited reduced islet vascularization and nascent endocrine cells in the ductal region. In addition, decreased ERK phosphorylation was observed in fetal MIPβ1KO pancreata. The expression of transcription factors needed for beta-cell development was unchanged in fetal MIPβ1KO pancreata. The findings from this study demonstrate that β1 integrin signaling is required during a transition-specific window in the developing beta-cell to maintain islet mass and vascularization.

Glycaemic control in diabetic rats treated with islet transplantation using plasma combined with hydroxypropylmethyl cellulose hydrogel

Islet transplantation is one of the most efficient cell therapies used in clinics and could treat a large proportion of patients with diabetes. However, it is limited by the high requirement of pancreas necessary to provide the sufficient surviving islet mass in the hepatic tissue and restore normoglycaemia. Reduction in organ procurement requirements could be achieved by extrahepatic transplantation using a biomaterial that enhances islet survival and function. We report a plasma-supplemented hydroxypropyl methylcellulose (HPMC) hydrogel, engineered specifically using a newly developed technique for intra-omental islet infusion, known as hOMING (h-Omental Matrix Islet filliNG). The HPMC hydrogel delivered islets with better performance than that of the classical intrahepatic infusion. After the validation of the HPMC suitability for islets in vivo and in vitro, plasma supplementation modified the rheological properties of HPMC without affecting its applicability with hOMING. The biomaterial association was proven to be more efficient both in vitro and in vivo, with better islet viability and function than that of the current clinical intrahepatic delivery technique. Indeed, when the islet mass was decreased by 25% or 35%, glycaemia control was observed in the group of plasma-supplemented hydrogels, whereas no regulation was observed in the hepatic group. Plasma gelation, observed immediately post infusion, decreased anoïkis and promoted vascularisation. To conclude, the threshold mass for islet transplantation could be decreased using HPMC-Plasma combined with the hOMING technique. The simplicity of the hOMING technique and the already validated use of its components could facilitate its transfer to clinics. Statement of significanceOne of the major limitations for the broad deployment of current cell therapy for brittle type 1 diabetes is the islets’ destruction during the transplantation process. Retrieved from their natural environment, the islets are grafted into a foreign tissue, which triggers massive cell loss.It is mandatory to provide the islets with an 3D environment specifically designed for promoting isletimplantation to improve cell therapy outcomes. For this aim, we combined HPMC and plasma.HPMC provides suitable rheological properties to the plasma to be injectable and be maintained in the omentum. Afterwards, the plasma polymerises around the graft in vivo, thereby allowing their optimal integration into their transplantation site.As a result, the islet mass required to obtain glycaemic control was reduced by 35%.

Regulation of c-Jun NH2-Terminal Kinase for Islet Transplantation.

Islet transplantation has been demonstrated to provide superior glycemic control with reduced glucose lability and hypoglycemic events compared with standard insulin therapy. However, the insulin independence rate after islet transplantation from one donor pancreas has remained low. The low frequency of islet grafting is dependent on poor islet recovery from donors and early islet loss during the first hours following grafting. The reduction in islet mass during pancreas preservation, islet isolation, and islet transplantation leads to β-cell death by apoptosis and the prerecruitment of intracellular death signaling pathways, such as c-Jun NH2-terminal kinase (JNK), which is one of the stress groups of mitogen-activated protein kinases (MAPKs). In this review, we show some of the most recent contributions to the advancement of knowledge of the JNK pathway and several possibilities for the treatment of diabetes using JNK inhibitors.

Impact of glucocorticoid treatment before pregnancy on glucose homeostasis of offspring exposed to glucocorticoid in adult life

AimsTo explore the impact of GC administration periconceptionally on the glucose metabolism of adult offspring (male and female) and whether this periconception exposure might influence the metabolic outcomes when the offspring are also treated with dexamethasone in adult life. Materials and methodsRats received a daily injection of dexamethasone (1 mg/kg, body mass) or saline solution (1 mL/kg body mass) for 7 consecutive days prior became pregnant. Male and female offspring had glucose homeostasis assessed at 3- and 6-month-old and after dexamethasone treatment (1 mg/kg, body mass) or vehicle for 5 consecutive days. Then, murinometric, functional, biochemical, and histomorphometric analyses were performed. Key findingsMale and female offspring born from rats treated with GC prior to becoming pregnant had none of the murinometric and metabolic outcomes (i.e., body mass, food intake, blood glucose, plasma triacylglycerol, and glucose tolerance) changed up to 6-month-old. None of the expected diabetogenic effects caused by dexamethasone treatment at 6-month of age (i.e., elevation in fasting blood glucose, plasma insulin, triacylglycerol, and albumin, glucose intolerance, insulin insensitivity, augmentation in hepatic glycogen content, and increase in pancreatic islet mass) was observed in offspring born from rats treated with dexamethasone in the prepregnancy period. However, periconceptional exposure to GC predisposed the offspring of both sexes to a higher prevalence of augmented fed blood glucose values. SignificanceThese results give validity for the use of GC as anti-inflammatory purposes in this critical periconceptional period, but highlight the importance to consider all parental habits when interpreting adult outcomes.

On the dynamics of the human endocrine pancreas and potential consequences for the development of type 1 diabetes

Little is known about the human islet life span, and beta-cell neogenesis is generally considered rare in adults. However, based on available data on beta-cell proliferation, calculations can be made suggesting that the dynamics of the endocrine pancreas is considerable even during adulthood, with islet neogenesis and a sustained increase in size of already formed islets. Islet-associated hemorrhages, frequently observed in most mammals including humans, could account for a considerable loss of islet parenchyma balancing the constant beta-cell proliferation. Notably, in subjects with type 1 diabetes, periductal accumulation of leukocytes and fibrosis is frequently observed, findings that are likely to negatively affect islet neogenesis from endocrine progenitor cells present in the periductal area. Impaired neogenesis would disrupt the balance, result in loss of islet mass, and eventually lead to beta-cell deficiency and compromised glucose metabolism, with increased islet workload and blood perfusion of remaining islets. These changes would impose initiation of a vicious circle further increasing the frequency of vascular events and hemorrhages within remaining islets until the patient eventually loses all beta-cells and becomes c-peptide negative.

Utility of arginine stimulation testing in preoperative assessment of children undergoing total pancreatectomy with islet autotransplantation.

Metabolic outcomes after total pancreatectomy with islet autotransplantation (TPIAT) are influenced by the islet mass transplanted. Preclinical and clinical studies indicate that insulin and C-peptide levels measured after intravenous administration of the beta cell secretagogue arginine can be used to estimate the available islet mass. We sought to determine if preoperative arginine stimulation test (AST) results predicted transplanted islet mass and metabolic outcomes in pediatric patients undergoing TPIAT. We evaluated the association of preoperative C-peptide and insulin responses to AST with islet isolation metrics using linear regression, and with postoperative insulin independence using logistic regression. Twenty-six TPIAT patients underwent preoperative AST from 2015 to 2018. The acute C-peptide response to arginine (ACRarg) was correlated with isolated islet equivalents (IEQ; r=0.59, P=0.002) and islet number (IPN; r=0.48, P=0.013). The acute insulin response to arginine (AIRarg) was not significantly correlated with IEQ (r=0.38, P=0.095) or IPN (r=0.41, P=0.071). Neither ACRarg nor AIRarg was associated with insulin use at 6months postoperatively. Preoperative C-peptide response to arginine correlates with islet mass available for transplant in pediatric TPIAT patients. AST represents an additional tool before autotransplant to provide counseling on likely islet mass and to inform quality improvements of islet isolation techniques.

2145-P: Notch1 Has Important Role in Beta-Cell Mass Determination and Development of Diabetes

Notch1 signaling is an important regulator of embryonic development and cell fate decision. Notch1 signaling is also expressed in adult pancreas but there is no study on the role of Notch1 in glucose homeostasis and islet mass in adult pancreas. Here, we investigated the role of Notch1 in glucose homeostasis and islet mass using Notch1 Antisense Transgenic (NAS) mice. We conducted an IPGTT and IPITT in 8-week-old male NAS mice and C57BL/6 mice (control mice). Insulin secretion capacity was measured by GSIS and perifusion. Morphology of pancreatic islet and β cell was conducted in two groups and transcription factors were evaluated. NAS mice showed higher glucose levels and lower insulin secretion in IPGTT. There was no significant difference in insulin resistance. Total islet and β cell masses were decreased in NAS mice. The number of large islets (≥ 250 μm) decreased while that of small islet (&amp;lt; 250 μm) increased. Reduced insulin secretion was observed in GSIS and perifusion. Our study provides that Notch1 inhibition decreased insulin secretion without any alterations in insulin resistance and decreased islet and β cell mass in the adult pancreas in NAS model. It is thought that Notch1 inhibition early suppresses islet proliferation and induces differentiation of not fully proliferated small islets. In conclusion, the Notch signaling pathway may play an important role in beta cell mass and development of diabetes. Disclosure Y. Eom: None. K. Kim: None. B. Kim: None. Funding Korea Health Technology R&amp;D Project (A101711, HI14C2539); Korean Ministry of Health, Welfare, and Family Affairs

2147-P: Pancreatic Specific Deletion of NOS1AP Lowered Islet Mass

Objective: Nitric oxide synthase associated protein (NOS1AP) is an adaptor protein regulating neuronal functions through NMDAR/nNOS-dependent pathway. We previously reported that NOS1AP SNP rs12742393 was associated with type 2 diabetes. In the present study we aimed to investigate the effect of NOS1AP on beta-cell function. Methods: Pancreatic specific deletion (CKO) of NOS1AP mice were generated from floxed nos1ap (exon 3-5) mice and pdx1-cre mice. These mice were fed with either chow or high fat diet (HFD,60% kcal fat). Results: NOS1AP was highly expression in mouse islets. Deletion of NOS1AP in pancreas had no effect on food intake and body weight in mice either fed with chow and high fat diet. However, deletion of NOS1AP impaired glucose tolerance in HFD fed mice. The blood glucose levels at 15min and 30min were higher in CKO than in its littermate control mice. The AUC of IPGTT in CKO was 20% higher than that in control mice. Insulin levels in CKO mice were lower than that in control mice after glucose load (P&amp;lt;0.05). Similarly, glucose stimulated insulin secretion from islets isolated from CKO was decreased compared with that from control mice. Moreover, islet mass in mice with pancreatic NOS1AP deletion decreased significantly (P&amp;lt;0.05). BrdU staining showed that the BrdU positive cells in islets were lower in NOS1AP CKO mice than in control mice. Overexpression of NOS1AP protected beta-cells from high levels of glucose and FFA (palmitate)-induced cell death. Conclusion: Our data demonstrate that NOS1AP plays a role in regulating beta-cell function and mass through regulation of cell proliferation and cytoprotection. Disclosure C. Wang: None. Q. Mao: None. Q. Li: None. W. Jia: None. Funding National Natural Science Foundation of China (81670707)

108-OR: Inhibition of SerpinB13 Stimulates Beta-Cell Development via Notch Signaling Pathway

Methods for repopulating the pancreas with new insulin-producing cells have strong potential for therapy in diabetes. Recently, we have found that inhibition of serpinB13 - a protease inhibitor of cathepsin L (catL) - with mAb in mouse embryos leads to a robust increase in the number of pancreatic Ngn3+ progenitor cells, significant expansion of islet mass, and improved resistance to severe diabetes in adulthood. To unveil the molecular mechanism of the augmented Ngn3+ cell response following inhibition of serpinB13 during gestation, we focused on the Notch communication system - a critical signaling pathway for pancreatic development. We found that serpinB13 is expressed and secreted by epithelial cells in murine embryonic pancreases. Moreover, in vivo and in vitro inhibition of serpinB13 during embryogenesis caused protease-dependent cleavage of the extracellular domain of Notch1 receptor in the pancreas (p&amp;lt;0.0001). This partial loss of the extracellular Notch was followed by decreased translocation to the nucleus of active Notch intracellular domain (aNICD), a fragment of Notch that is critical for restraining endocrine cell development. Finally, embryonic pancreases of mice with genetic deficiency of catL had significantly fewer Ngn3+ cells compared with wild type controls. Together, our data point to a novel function of serpinB13 in maintaining Notch receptor-mediated repression of pancreatic endocrine progenitors. Consequently, the perturbation of this effect of serpinB13 enables protease activity to partially dismantle Notch signaling, thereby allowing for more efficient development of Ngn3+ progenitors cells and a subsequent increase in islet mass. Disclosure Y. Kryvalap: None. C. Hendrickson: None. J. Czyzyk: None. Funding American Diabetes Association (1-17-ICTS-083 to J.C.)

337-LB: Combined Oral Administration of GABA and GLP-1 Promotes Human ß-Cell Proliferation and Reduces Apoptosis

Gamma-aminobutyric acid (GABA) and glucagon-like peptide-1 (GLP-1) improve rodent β cell survival and function. In human β cells, GABA exerts stimulatory effects on proliferation and anti-apoptotic effects, whereas GLP-1-based drugs have only limited effects on proliferation. We previously demonstrated that GABA and sitagliptin, a dipeptidyl peptidase-4 inhibitor which increases endogenous GLP-1 levels, mediated a synergistic β cell protective effect in mice islets. However, it remains unclear whether this combination has similar effects on human β cells. To address this question, we transplanted a suboptimal mass of human islets into immunodeficient NOD-scid-gamma mice with streptozotocin-induced diabetes, and then treated them with GABA, sitagliptin, or both. The oral administration of either GABA or sitagliptin ameliorated blood glucose levels, increased transplanted human β cell mass and plasma human insulin levels. However, combined administration of the drugs generated significantly superior results in all these responses, as compared to monotherapy. Proliferation and/or regeneration, improved by the combination, were demonstrated by increased Ki67+, pancreatic and duodenal homeobox-1 (PDX-1+), or Nkx6.1+ β cell numbers. Protection against apoptosis was also significantly improved by the combined therapy. GABA and GLP-1 in combination notably increased Akt phosphorylation and insulin secretion, as well as the expression of sirtuin-1 and α-Klotho, both of which have been reported to have protective effects on β-cells. Our study indicates that combined use of GABA and sitagliptin produced greater therapeutic benefits, which was likely due to an enhancement of β cell proliferation and a decrease of apoptosis. Disclosure W. Liu: None. T. Jin: None. Q. Wang: None. Funding National Natural Science Foundation of China (81570518, 81630020); JDRF (2-SRA-2017-64-G-R to Q.W., T.J.), (2-SRA-2015-64 to Q.W., T.J.); Canadian Diabetes Association (OG-3-13-4066 to Q.W.)

2128-P: Exploring the Role of Autophagy in Pancreatic Alpha Cells

Autophagy is known to play a pivotal role in intracellular quality control through the degradation of subcellular damaged organelles and components. Whereas autophagy is essential for maintaining β-cell function in pancreatic islets, it remains unclear as to how the cellular autophagy affects the homeostasis and function of glucagon-secreting α cells. To analyze the role of autophagy in α cells, we generated a mutant mouse model lacking Atg7, a key molecule for autophagosome formation, specifically in α cells. Histological analysis demonstrated an increased number of glucagon-positive cells, with a multilayered structure, in the islets under Atg7 deficiency, although metabolic profiles, such as body weight, blood glucose and plasma glucagon levels, were comparable between Atg7-deficient mice and control littermates. Consistent with our previous findings that Atg7 deficiency suppressed β-cell proliferation, cellular proliferation was suppressed in Atg7-deficient α cells. These findings suggest that α-cell autophagy plays a role in maintaining α-cell mass and normal islet architecture, but appears to be dispensable for metabolic homeostasis. Disclosure M. Himuro: None. T. Miyatsuka: None. L. Suzuki: None. M. Miura: None. T. Katahira: None. H. Goto: None. Y. Nishida: None. S. Sasaki: None. Y. Fujitani: Research Support; Self; Takeda Pharmaceutical Company Limited. H. Watada: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Kissei Pharmaceutical Co., Ltd., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Pfizer Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Sanofi, Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. Other Relationship; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Kowa Pharmaceutical Europe Co. Ltd., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Ono Pharmaceutical Co., Ltd., Sanwa Chemical Industry Co. Ltd., Takeda Pharmaceutical Company Limited. Funding Japan Society for the Promotion of Science

191-LB: Artesunate Treatment Improves Hyperglycemia in a Rodent Model of Gestational Glucose Intolerance without Increasing Beta-Cell Mass

Gestational diabetes (GDM) has been associated with a failure to adequately increase maternal β-cell mass (BCM) during pregnancy. The anti-malarial drugs, artemisinins, have been shown to improve glucose homeostasis in rodent models of diabetes by increasing BCM. Our objective was to reverse BCM deficiency and impaired glucose tolerance in a mouse model of GDM using the artemisinin artesunate. In order to produce glucose intolerance in pregnancy, female mice (F0) were fed a low protein (8%, LP) diet during gestation and lactation, and offspring weaned onto control (20%, C) diet. At 6-10 weeks age female offspring (F1) were time-mated and given C diet throughout pregnancy. Artesunate was administered via drinking water from gestational day (GD) 0.5-6.5. We previously found glucose intolerance, mimicking aspects of GDM, in LP-exposed offspring at GD18 when compared to animals fed a C diet. Thus, glucose tolerance tests (GTT) were performed at GD18 or for non-pregnant age-matched animals. Subsequently, pancreata were removed for fluorescence immunohistochemistry to assess histology. Artesunate-treated LP females had a lower area under the curve during a GTT (389.9±52.3mmol/L*min) compared to non-treated LP females at GD18 (726.6±56.9mmol/L*min, p&amp;lt;0.01). Furthermore, artesunate-treated LP females had improved glucose homeostasis at 5min (9.1 vs. 18.9mmol/L), 15min (11.6 vs. 20.9mmol/L) and 30min (10.2 vs. 19.2mmol/L) during the GTT compared to non-treated LP females at GD18 (p&amp;lt;0.05). There were no significant differences in BCM, alpha-cell mass, or mean islet size between LP and artesunate-treated LP females at GD18. The results show that artesunate-treated LP pregnant females had significantly improved glucose homeostasis compared to untreated LP mice at GD18. However, the improved glucose tolerance was not associated with increased BCM or changes in endocrine pancreas morphology. Disclosure S. Szlapinski: None. D.J. Hill: None. Funding St. Joseph's Health Care Foundation; Ontario Graduate Scholarship