Β-cell Proliferation Research Articles

The Role of Prolactin Signalling in Beta-cell Adaptation to Metabolic Stressors

The ability of β cells. to adapt to metabolic stressors is important in preventing diabetes. Our lab has previously shown that prolactin receptor (Prlr) signalling is required for β-cell adaptation to insulin resistance of pregnancy, mainly by regulating β-cell proliferation. Whether Prlr signalling is important for β-cell adaptation to physiologic stresses other than pregnancy is unknown. This study aimed to determine whether Prlr signalling is required for β-cell adaptation to the cumulative stresses of pregnancies followed by exposure to a high fat diet (HFD), a commonly observed scenario in humans.

Molecular dynamics study of tropical calcific pancreatitis (TCP) associated calcium-sensing receptor single nucleotide variation.

Tropical Calcific Pancreatitis (TCP) is a chronic non-alcoholic pancreatitis characterised by extensive calcification. The disease usually appears at a younger age and is more common in tropical regions. This disease’s progression can lead to pancreatic diabetes, which can subsequently lead to pancreatic cancer. The CASR gene encodes a calcium-sensing receptor (CaSR), which is a GPCR protein of class C. It is expressed in the islets of Langerhans, the parathyroid gland, and other tissues. It primarily detects small gradients in circulating calcium concentrations and couples this information to intracellular signalling, which helps to regulate PTH (parathyroid hormone) secretion and mineral ion homeostasis. From co-leading insulin release, CaSR modulates ductal HCO3− secretion, Ca2+ concentration, cell-cell communication, β-cell proliferation, and intracellular Ca2+ release. In pancreatic cancer, the CaSR limits cell proliferation. TCP-related four novel missense mutations P163R, I427S, D433H and V477A, found in CaSR extracellular domain (ECD) protein, which were reported in the mutTCPdb Database (https://lms.snu.edu.in/mutTCPDB/index.php). P163R mutation occurs in ligand-binding domain 1 (LBD-1) of the CaSR ECD. To investigate the influence of these variations on protein function and structural activity multiple in-silico prediction techniques such as SIFT, PolyPhen, CADD scores, and other methods have been utilized. A 500 ns molecular dynamic simulation was performed on the CaSR ECD crystal structure and the corresponding mutated models. Furthermore, Principal Component Analysis (PCA) and Essential Dynamics analysis were used to forecast collective motions, thermodynamic stabilities, and the critical subspace crucial to CaSR functions. The results of molecular dynamic simulations showed that the mutations P163R, I427S, D433H, and V477A caused conformational changes and decreased the stability of protein structures. This study also demonstrates the significance of TCP associated mutations. As a result of our findings, we hypothesised that the investigated mutations may have an effect on the protein’s structure and ability to interact with other molecules, which may be related to the protein’s functional impairment.

Single-Cell RNA Sequencing Reveals a Role for Reactive Oxygen Species and Peroxiredoxins in Fatty Acid-Induced Rat β-Cell Proliferation.

The functional mass of insulin-secreting pancreatic β-cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing β-cells. Type 2 diabetes appears when these compensatory mechanisms fail. Nutrients including glucose and fatty acids are important contributors to the β-cell compensatory response, but their underlying mechanisms of action remain poorly understood. We investigated the transcriptional mechanisms of β-cell proliferation in response to fatty acids. Isolated rat islets were exposed to 16.7 mmol/L glucose with or without 0.5 mmol/L oleate (C18:1) or palmitate (C16:0) for 48 h. The islet transcriptome was assessed by single-cell RNA sequencing. β-Cell proliferation was measured by flow cytometry. Unsupervised clustering of pooled β-cells identified different subclusters, including proliferating β-cells. β-Cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved in energy metabolism and mitochondrial activity. Comparison of proliferating versus nonproliferating β-cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, N-acetyl cysteine and the peroxiredoxin inhibitor conoidin A both blocked oleate-induced β-cell proliferation. Our study reveals a key role for ROS signaling through peroxiredoxin activation in oleate-induced β-cell proliferation.

E2F1 transcription factor mediates a link between fat and islets to promote β cell proliferation in response to acute insulin resistance.

SUMMARYPrevention or amelioration of declining β cell mass is a potential strategy to cure diabetes. Here, we report the pathways utilized by β cells to robustly replicate in response to acute insulin resistance induced by S961, a pharmacological insulin receptor antagonist. Interestingly, pathways that include CENP-A and the transcription factor E2F1 that are independent of insulin signaling and its substrates appeared to mediate S961-induced β cell multiplication. Consistently, pharmacological inhibition of E2F1 blocks β-cell proliferation in S961-injected mice. Serum from S961-treated mice recapitulates replication of β cells in mouse and human islets in an E2F1-dependent manner. Co-culture of islets with adipocytes isolated from S961-treated mice enables β cells to duplicate, while E2F1 inhibition limits their growth even in the presence of adipocytes. These data suggest insulin resistance-induced proliferative signals from adipocytes activate E2F1, a potential therapeutic target, to promote β cell compensation.

Betatrophin and Insulin Resistance.

Betatrophin (angiopoietin-like protein 8 (ANGPTL8)) is a hormone that was recently discovered in the human liver. Multiple homologous sequences have been detected in mammalian liver, white adipose, and brown adipose tissues. Betatrophin is crucial for the development of type 2 diabetes (T2D), insulin resistance, and lipid metabolism. Similar to the intake of insulin, thyroid hormones, irisin, and calories, betatrophin expression in the organism is usually attributed to energy consumption or heat generation. It can mediate the activity of lipoprotein lipase (LPL), which is the key enzyme of lipoprotein lipolysis. Due to its association with metabolic markers and the roles of glucose and lipid, the physiological function of betatrophin in glucose homeostasis and lipid metabolism can be more comprehensively understood. Betatrophin was also shown to facilitate pancreatic β-cell proliferation in a mouse model of insulin resistance. There are also reports that demonstrate that betatrophin regulates triglycerides (TGs) in the liver. Therefore, the process of regulating the physiological function by betatrophin is complicated, and its exact biological significance remains elusive. This study provides a comprehensive review of the current research, and it discusses the possible physiological functions of betatrophin, and specifically the mechanism of betatrophin in regulating blood glucose and blood lipids.

γ-aminobutyric acid modulates α-cell hyperplasia but not β-cell regeneration induced by glucagon receptor antagonism in type 1 diabetic mice.

To investigate whether treatment with γ-aminobutyric acid (GABA) alone or in combination with glucagon receptor (GCGR) monoclonal antibody (mAb) exerted beneficial effects on β-cell mass and α-cell mass, and to explore the origins of the regenerated β-cells in mice with type 1 diabetes (T1D). Streptozotocin (STZ)-induced T1D mice were treated with intraperitoneal injection of GABA (250μg/kg per day) and/or REMD 2.59 (a GCGR mAb, 5mg/kg per week), or IgG dissolved in PBS for 8weeks. Plasma hormone levels and islet cell morphology were evaluated by ELISA and immunofluorescence, respectively. The origins of the regenerated β-cells were analyzed by double-immunostaining, α-cell lineage-tracing and BrdU-tracing studies. After the 8-week treatment, GABA or GCGR mAb alone or in combination ameliorated hyperglycemia in STZ-induced T1D mice. GCGR mAb upregulated plasma insulin level and increased β-cell mass, and GABA appeared to have similar effects in T1D mice. However, combination treatment did not reveal any additive or synergistic effect. Interestingly, the GCGR mAb-induced increment of plasma glucagon level and α-cell mass was attenuated by the combined treatment of GABA. In addition, duct-derived β-cell neogenesis and α-to-β cell conversion but not β-cell proliferation contributed to the increased β-cell mass in T1D mice. These results suggested that GABA attenuated α-cell hyperplasia but did not potentiates β-cell regeneration induced by GCGR mAb in T1D mice. Our findings provide novel insights into a combination treatment strategy for β-cell regeneration in T1D.

Therapeutic potential of vasoactive intestinal peptide and its receptor VPAC2 in type 2 diabetes.

Owing to the increasing prevalence of type 2 diabetes, the development of novel hypoglycemic drugs has become a research hotspot, with the ultimate goal of developing therapeutic drugs that stimulate glucose-induced insulin secretion without inducing hypoglycemia. Vasoactive intestinal peptide (VIP), a 28-amino-acid peptide, can stimulate glucose-dependent insulin secretion, particularly by binding to VPAC2 receptors. VIP also promotes islet β-cell proliferation through the forkhead box M1 pathway, but the specific molecular mechanism remains to be studied. The clinical application of VIP is limited because of its short half-life and wide distribution in the human body. Based on the binding properties of VIP and VPAC2 receptors, VPAC2-selective agonists have been developed to serve as novel hypoglycemic drugs. This review summarizes the physiological significance of VIP in glucose homeostasis and the potential therapeutic value of VPAC2-selective agonists in type 2 diabetes.

Critical Role for Macrophages in the Developmental Programming of Pancreatic β-Cell Area in Offspring of Hypertensive Pregnancies.

Preeclampsia is a pregnancy-specific complication with long-term negative outcomes for offspring, including increased susceptibility to type 2 diabetes (T2D) in adulthood. In a rat reduced uteroplacental perfusion pressure (RUPP) model of chronic placental ischemia, maternal hypertension in conjunction with intrauterine growth restriction mimicked aspects of preeclampsia and resulted in female embryonic day 19 (e19) offspring with reduced β-cell area and increased β-cell apoptosis compared with offspring of sham pregnancies. Decreased pancreatic β-cell area persisted to postnatal day 13 (PD13) in females and could influence whether T2D developed in adulthood. Macrophage changes also occurred in islets in T2D. Therefore, we hypothesized that macrophages are crucial to reduction in pancreatic β-cell area in female offspring after chronic placental ischemia. Macrophage marker CD68 mRNA expression was significantly elevated in e19 and PD13 islets isolated from female RUPP offspring compared with sham. Postnatal injections of clodronate liposomes into female RUPP and sham offspring on PD2 and PD9 significantly depleted macrophages compared with injections of control liposomes. Depletion of macrophages rescued reduced β-cell area and increased β-cell proliferation and size in RUPP offspring. Our studies suggest that the presence of macrophages is important for reduced β-cell area in female RUPP offspring and changes in macrophages could contribute to development of T2D in adulthood.

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.

Long Noncoding RNA-5657 Inhibits the Proliferation of Pancreatic Cells and Insulin Secretion in Gestational Diabetes by Inhibiting the NADPH Oxidase 4 (NOX4)

Multiple comorbidities are observed during pregnancy and there are many challenges for pregnant women with a previous history of diabetes. Pancreatic β-cell dysfunction is associated with gestational diabetes mellitus (GDM). This study intends to explore LncRNA-5657’s effect on proapoptotic cell proliferation and insulin secretion in gestational diabetes. Real-time quantitative fluorescence PCR measured LncRNA-5657 expression in GDM patient placental tissues along with analysis of cell activity, proliferation and insulin secretion by MTT assay, BrdU staining and ELISA. The relation between LncRNA-5657 and NOX4 was assessed by dual luciferase reporter assay. LncRNA-5657 was overexpressed in placental tissue of GDM patients compared to normal pregnant women. LncRNA-5657 overexpression promoted cell proliferation and insulin secretion, which was reversed by LncRNA-5657 downregulation. In addition, LncRNA-5657 negatively regulated NOX4 expression. Overexpression of NOX4 abolished the effect of LncRNA-5657 mimics on pancreatic β-cells, and knockdown of LncRNA-5657 promoted insulin secretion by upregulating NOX4 expression. In conclusion, LncRNA-5657 inhibits insulin secretion and β-cell proliferation by targeting NOX4, indicating that LncRNA-5657 might be a novel target for the treatment of GDM.

Regeneration of β-cells of the islet apparatus of the pancreas. Literature review

Diabetes of both type 1 and type 2 is characterized by a progressive loss of β-cell mass, which contributes to the disruption of glucose homeostasis. The optimal antidiabetic therapy would be simple replacement of lost cells, but at present, many researchers have shown that the pancreas (PZ) of adults has a limited regenerative potential. In this regard, significant efforts of researchers are directed to methods of inducing the proliferation of β-cells, stimulating the formation of β-cells from alternative endogenous sources and/or the generation of β-cells from pluripotent stem cells. Factors that regulate β-cell regeneration under physiological or pathological conditions, such as mediators, transcription factors, signaling pathways and potential pharmaceuticals, are also being intensively studied. In this review, we consider recent scientific studies carried out in the field of studying the development and regeneration of insulin-producing cells obtained from exogenous and endogenous sources and their use in the treatment of diabetes. The literature search while writing this review was carried out using the databases of the RSIC, CyberLeninka, Scopus, Web of Science, MedLine, PubMed for the period from 2005 to 2021. using the following keywords: diabetes mellitus, pancreas, regeneration, β-cells, stem cells, diabetes therapy.

The predictive diagnosis for ANGPTL8 level in women with T2DM

Background: The metabolic syndrome including type 2 diabetes mellitus(T2DM), insulin resistance(IR), obese, hypertension, and dyslipidemia, is an increasing health burden. In the last decade, various metabolic hormones have been noticed to significantly role obese and associated complications. for example of this, the adipokines or hepatokines secreted proteins from hepatocyte and adipocyteare correlate with metabolic syndrome. Angiopoietin-like protein 8 (ANGPTL8) has a novel hepatokine or adipokine, which is mostly expressed in the adipose tissue, and liver stimulates the β-cell proliferation as a pancreatic in an insulin resistance state. It's a recently discovered the endocrine regulator associated with glucose homeostasis, IR, and metabolism of lipid. Materials and Methods: Ninety women participated in this study, comprising sixty patients with diabetes mellitus type-2 and thirty healthy (sex and age - matched) as a control group. Enzyme linked immune sorbent assay (ELISA) was used to assess ANGPTL8 and insulin. Standard procedures were used to measure fasting blood glucose, lipid profile, and glycated hemoglobin (HBA1c) in a certified laboratory. Results: Diabetes patients had higher levels of ANGPTL8 than the control group. The blood ANGPTL8 level in the Type-2 diabetes group varied from 4432.89±1171.09 pg/ml, while the control group's level was 2892.11±537.91pg/dl (p<0.0001).

An Overfeeding-Induced Obesity Mouse Model Reveals Necessity for Sin3a in Postnatal Peak β-Cell Mass Acquisition.

The increase of functional β-cell mass is paramount to maintaining glucose homeostasis in the setting of systemic insulin resistance and/or augmented metabolic load. Understanding compensatory mechanisms that allow β-cell mass adaptation may allow for the discovery of therapeutically actionable control nodes. In this study, we report the rapid and robust β-cell hyperplasic effect in a mouse model of overfeeding-induced obesity (OIO) based on direct gastric caloric infusion. By performing RNA sequencing in islets isolated from OIO mice, we identified Sin3a as a novel transcriptional regulator of β-cell mass adaptation. β-Cell-specific Sin3a knockout animals showed profound diabetes due to defective acquisition of postnatal β-cell mass. These findings reveal a novel regulatory pathway in β-cell proliferation and validate OIO as a model for discovery of other mechanistic determinants of β-cell adaptation.

A Chinese medical nutrition therapy diet accompanied by intermittent energy restriction alleviates type 2 diabetes by enhancing pancreatic islet function and regulating gut microbiota composition

Medicine food homologous (MFH) plants and wholegrains have gained increasing attention for prevention and treatment of type 2 diabetes (T2D). Substantial evidence supports the effectiveness of intermittent energy restriction (IER) in T2D management. However, there are few studies that report intermittent use of a low-calorie pre-prepared food including MFH plants and wholegrains in T2D. The aim of this study was to investigate the effects of Chinese Medical Nutrition Therapy (CMNT), a MFH plants and wholegrains diet accompanied by IER, on glycemic control and potential mechanism. Ten-week-old diabetic db/db mice were randomly divided into CMNT group (feeding low-calorie mouse CMNT diet in day 1–4 and ad libitum regular chow for up to 7 days per cycle) and control group (ad libitum access to regular chow). The results showed that CMNT reduced fasting blood glucose, improved glucose tolerance with higher insulin secretion, attenuated macrophage infiltration, promoted β-cell proliferation of pancreatic islets, and increased diabetes-improving microbiota (Bacteroides, Rikenellaceae_RC9_gut_group and Coprococcus_1) in db/db mice. Additionally, we performed a pilot study evaluating CMNT in thirty-nine T2D patients without obesity. Participants with T2D randomly assigned to two groups: CMNT group (receiving a consecutive 5-day low-calorie human CMNT diet with 10 days of habitual eating per cycle for 90 days) and control group (continuing on a normal diet). We observed an improvement in glycemic control in CMNT group with significant reduction in HbA1c, fasting glucose, 2 h postprandial blood glucose but control group were not affected. After CMNT intervention, the abundance of the phylum Bacteroidetes, and genus Bacteroides, Parabacteroides and Roseburia were significantly higher than baseline in T2D patient, which were closely associated with glycemic control. These findings suggested that CMNT is a promising nutritional intervention approach in diabetes management.

Evaluation of the Effects of Harmine on β-cell Function and Proliferation in Standardized Human Islets Using 3D High-Content Confocal Imaging and Automated Analysis.

Restoration of β-cell mass through the induction of proliferation represents an attractive therapeutic approach for the treatment of diabetes. However, intact and dispersed primary islets suffer from rapidly deteriorating viability and function ex vivo, posing a significant challenge for their experimental use in proliferation studies. Here, we describe a novel method for the assessment of compound effects on β-cell proliferation and count using reaggregated primary human islets, or islet microtissues (MTs), which display homogeneous size and tissue architecture as well as robust and stable functionality and viability for 4 weeks in culture. We utilized this platform to evaluate the dose-dependent short- and long-term effects of harmine on β-cell proliferation and function. Following compound treatment and EdU incorporation, islet MTs were stained and confocal-imaged for DAPI (nuclear marker), NKX6.1 (β-cell marker), and EdU (proliferation marker), allowing automated 3D-analysis of number of total cells, β-cells, and proliferating β- and non-β-cells per islet MT. In parallel, insulin secretion, intracellular insulin and ATP contents, and Caspase 3/7 activity were analyzed to obtain a comprehensive overview of islet MT function and viability. We observed that 4-day harmine treatment increased β- and non-β-cell proliferation, NKX6.1 expression, and basal and stimulated insulin secretion in a dose-dependent manner, while fold-stimulation of secretion peaked at intermediate harmine doses. Interestingly, 15-day harmine treatment led to a general reduction in harmine’s proliferative effects as well as altered dose-dependent trends. The described methodology provides a unique tool for in vitro high-throughput evaluation of short- and long-term changes in human β-cell proliferation, count and fraction along with a variety of functional parameters, in a representative 3D human islet model.

Association of protein function-altering variants with cardiometabolic traits: the strong heart study

Clinical and biomarker phenotypic associations for carriers of protein function-altering variants may help to elucidate gene function and health effects in populations. We genotyped 1127 Strong Heart Family Study participants for protein function-altering single nucleotide variants (SNV) and indels selected from a low coverage whole exome sequencing of American Indians. We tested the association of each SNV/indel with 35 cardiometabolic traits. Among 1206 variants (average minor allele count = 20, range of 1 to 1064), ~ 43% were not present in publicly available repositories. We identified seven SNV-trait significant associations including a missense SNV at ABCA10 (rs779392624, p = 8 × 10–9) associated with fasting triglycerides, which gene product is involved in macrophage lipid homeostasis. Among non-diabetic individuals, missense SNVs at four genes were associated with fasting insulin adjusted for BMI (PHIL, chr6:79,650,711, p = 2.1 × 10–6; TRPM3, rs760461668, p = 5 × 10–8; SPTY2D1, rs756851199, p = 1.6 × 10–8; and TSPO, rs566547284, p = 2.4 × 10–6). PHIL encoded protein is involved in pancreatic β-cell proliferation and survival, and TRPM3 protein mediates calcium signaling in pancreatic β-cells in response to glucose. A genetic risk score combining increasing insulin risk alleles of these four genes was associated with 53% (95% confidence interval 1.09, 2.15) increased odds of incident diabetes and 83% (95% confidence interval 1.35, 2.48) increased odds of impaired fasting glucose at follow-up. Our study uncovered novel gene-trait associations through the study of protein-coding variants and demonstrates the advantages of association screenings targeting diverse and high-risk populations to study variants absent in publicly available repositories.

248-LB: Nrf2 Is Essential for Expanding Neonatal Pancreatic Beta-Cell Mass

Patients with both major forms of diabetes suffer from insufficient functional β-cell mass. Moreover, the sharp increase in the annual incidence of both type 1 (1.8%) and type 2 (4.8%) diabetes in the pediatric population in the last decades highlights the urgent need for discovering the mechanisms that regulate functional β-cell mass. β-cell proliferation, which reaches its maximal peak during postnatal stages is the major mechanism for increasing β-cell mass in early postnatal ages. We previously found that induction of the antioxidant regulator, Nrf2, is required for adaptive β-cell expansion under hypercaloric conditions via stimulation of β-cell proliferation, increased β-cell survival, and maintenance of β-cell identity in adult mice. Based on these observations, we hypothesize that Nrf2 regulates β-cell proliferation and survival at early stages of life and contribute to maintain normal β-cell mass later in life. Nrf2 expression levels were found to correlate with β-cell proliferation in wild type neonatal mice. Importantly, 28 day-old β-cell specific Nrf2 knockout mice (Ins1Cre-Nrf2lox/lox) exhibited 50% reduction in β-cell mass, 58% reduction in total islet number, 43% decrease in extra-large islets, 46% decrease in large islets and 53% decrease in extra small islets, compared to control mice. No changes in pancreas weight were observed. Additionally, depletion of Nrf2 in β-cells increased β-cell death in 7 (5.8-fold) and 14 (12.5-fold) day-old mice and attenuated β-cell proliferation in 1- and 7-day-old mice. Surprisingly, we also found a marked decrease in islet size in 1- and 7-day-old mice, suggesting that Nrf2 may affect β-cell proliferation, survival and/or development during embryonic stages. We conclude that Nrf2 is required for β-cell mass expansion in postnatal ages by controlling β-cell proliferation and survival. Disclosure S. Baumel-alterzon: None. A. Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. D. Scott: None.

203-OR: VGLL4 and MENIN Function as TEAD1 Corepressors to Block Pancreatic ß-Cell Proliferation

β-cell mass is significantly reduced especially in the late stage of diabetes. Hence, increasing β-cell proliferation to increase β-cell mass remains a desirable goal for diabetes treatment. The mechanisms that regulate β cell proliferation and concurrent maintenance of mature function is not well understood. Hippo-TEAD pathways regulated proliferation in many tissues. Our data show that TEAD1 (Hippo) pathway activities were significantly reduced in mature β cell. TEAD1 knockout results in the cell-autonomous increase of β cell proliferation, while loss of TAZ has not change in β cell proliferation, suggesting that TEAD1 corepressors, rather than coactivators, play a dominant role in β-cells. Improved Split-GFP system and yeast two-hybrid platform were also adapted to screen TEAD1 cofactors in β cells. Our study showed that MENIN can bind to TEAD1 directly and the pocket area of TEAD1 is critical for this binding. VGLL4 and MENIN work as TEAD1 corepressors to inhibit the expression of target genes in β-cells, including FZD7 and CCN2, which leads to a restriction of β cell proliferation. We also found that TEAD1 can regulate TEAD1, and this auto-inhibition contributes to TEAD1 pathway inhibition with β cell maturity. Our study sheds light on the mechanisms involved in β cell maintenance of mature function and proliferation and offer the mechanistic basis for modulating TEAD pathway to increase proliferation in β cells. Disclosure F.Li: None. R.Liu: None. V.Negi: None. P.Yang: None. J.K.Lee: None. M.Moulik: None. V.Yechoor: n/a.

201-OR: Evaluation of the Effects of Harmine on ß-Cell Function and Proliferation in Standardized Human Islets Using 3D High-Content Confocal Imaging and Automated Analysis

Restoration of β-cell mass by inducing proliferation of residual β-cells represents an attractive therapeutic approach for diabetes treatment. Native and dispersed primary human islets undergo rapid loss of viability and function ex vivo, complicating their use in proliferation studies. Here, we establish a novel method for evaluating compound effects on β-cell proliferation and count using reaggregated primary human islet microtissues (MTs) , of homogeneous size and architecture and that maintain stable viability and function for 4 weeks in culture. We utilized this platform to evaluate the effects of short- and long-term harmine treatment on β-cell proliferation and function in a dose-dependent manner. Following harmine treatment and EdU incorporation, islet MTs were stained in 3D for DAPI (nuclear marker) , NKX6.1 (β-cell marker) , and EdU (proliferation marker) , imaged on a high-content confocal microscope, and subjected to automated 3D analysis of numbers of total cells, β-cells, and proliferating β- and non-β-cells per islet MT. In parallel, assessment of insulin secretion, intracellular insulin and ATP contents, and Caspase 3/7 activity was performed for a comprehensive overview of islet MT function and viability. We observed that 4 days of harmine treatment increased β- and non-β-cell proliferation, NKX6.1 expression, and basal and stimulated insulin secretion in a dose-dependent manner, whereas fold-stimulation of secretion peaked at intermediate doses. Interestingly, 15-day harmine treatment led to a general reduction in proliferative effects as well as altered dose-dependent trends. The methodology presented here thus constitutes a unique tool for in vitro high-throughput evaluation of short- and long-term changes in human β-cell proliferation, count and fraction in parallel with a variety of functional parameters, in a representative and standardized 3D human islet microtissue model. Disclosure A.C.Title: None. B.Yesildag: None. M.Karsai: None. J.Mir-coll: None. O.Yavas grining: None. C.Rufer: None. S.Sonntag: None. F.Forschler: None. S.Jawurek: None. T.Klein: Employee; Boehringer Ingelheim International GmbH.

1375-P: Effects of Irisin Administration on Pancreatic Islets of Diabetic Mice

Irisin is a hormone secreted by skeletal muscle following exercise or excess of saturated fatty acids (SFAs) , able to improve metabolic homeostasis and promote energy expenditure. Serum irisin levels are reduced in type 2 diabetes (T2D) , while exogenous irisin administration improves glycemic control in diabetic mice. We have previously demonstrated that irisin protects human and rodent β-cells and pancreatic islets from SFAs-induced apoptosis, increases insulin biosynthesis and glucose-stimulated insulin secretion (GSIS) , and promotes β-cell proliferation, both in vitro and in vivo in healthy wild-type mice. Irisin may also restore the defective GSIS and reduce the high rate of apoptosis that are characteristic of islets from T2D patients. However, the β-cellular effects of irisin administration to diabetic mice are still unknown. 6 weeks-old C57Bl/6 mice (n = 8) were fed a high-fat diet (HFD, 60% of energy deriving from fat) for weeks and then intraperitoneally injected with streptozotocin (STZ, 100 mg/Kg) to induce diabetes. 4 standard diet (SD) -fed mice were used as control. HFD/STZ mice were then treated with 0.5 μg/g irisin (n = 4) or vehicle (n = 4) for 14 days. Glucose tolerance, glycemia, insulinemia, body weight, pancreatic islet architecture and function were assessed throughout the study. As expected, compared to SD mice, HFD/STZ mice showed higher glycemia and body weight, glucose intolerance, and reduced GSIS; in addition, HFD/STZ mice showed reduced islet volume (-78%) , β-cell area (-35%) , and insulin content (-60%) , while increased α-cell area (+54%) . Irisin administration significantly restored glycemia (-31%) , body weight (-13%) , glucose tolerance (-27%, AUC) , GSIS (+23%, AUC) , islet volume (+61%) , β-cell (+34%) and α-cell (-49%) areas, and insulin content (+36%) . Of note, irisin treatment induced a 9-fold increase in β-cell proliferation rate. These results show for the first time that irisin can restore β-cell functional mass when administered in vivo to diabetic mice. Disclosure N.Marrano: None. G.Biondi: None. A.Borrelli: None. L.Roberto: None. A.Cignarelli: None. S.Perrini: None. L.Laviola: Advisory Panel; Lilly Diabetes, Novo Nordisk, Roche Diabetes Care, Sanofi, Speaker's Bureau; A. Menarini Diagnostics, Abbott Diabetes, Medtronic, Terumo Corporation. F.Giorgino: Advisory Panel; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk, Consultant; Lilly Diabetes, Sanofi, Research Support; Lilly Diabetes, Roche Diabetes Care, Takeda Pharmaceutical Company Limited. A.Natalicchio: Consultant; Novo Nordisk, Other Relationship; AstraZeneca, Lilly Diabetes, Sanofi. Funding EU – ESF PON R&I 2014-20, AIM