Beta Cell Gene Expression Research Articles

Diabetes Risk Allele of Transcription Factor 7-like 2 (TCF7L2) Polymorphisms is Associated with Higher Glucagon-like Peptide 1 (GLP1) and Lower Insulin Secretion

BACKGROUND: The most influential susceptible gene associated with diabetes, transcription factor 7-like 2 (TCF7L2), has been observed in diverse populations. TCF7L2 influences type 2 diabetes risk through glucagon-like peptide 1 (GLP1) production. The presence of risk allele of TCF7L2 leads to the alteration of gene expression in pancreatic beta cells; however, how the mechanism is related with GLP1 remains unclear. This study was conducted to explore the variations of GLP1 increment and insulin secretion between individuals with and without diabetes risk allele of single nucleotide polymorphisms (SNPs) in TCF7L2.METHODS: A cross-sectional analytic study was conducted involving individuals subjects who harbored known variants of SNPs in the TCF7L2: heterozygote or mutant of rs12255372 (GT or TT), rs7903146 (CT or TT), rs10885406 (AG or GG); as well as control subjects with wild type of rs12255372 (GG), rs7903146 (CC), and rs10885406 (AA). Anthropometric parameters, blood glucose, insulin, and GLP1 were measured; and homeostasis model assessment-beta cell (HOMA-%B) index was calculated.RESULTS: The GLP1 increment response was higher in subjects carrying the diabetes risk allele (0.34±0.80 ng/mL) than those with the wild type (-0.04±0.57 ng/mL) (p=0.041). The HOMA-%B was reduced in subjects carrying the diabetes risk allele (71.64±24.72) than those with the wild type (103.23±68.00) (p=0.011). Among individuals carrying the diabetes risk allele, the likelihood of GLP1 increment with high response was twice as high (p=0.007), while the occurrence of low HOMA-%B was 1.47 more frequent (p=0.011).CONCLUSION: TCF7L2 polymorphisms were associated with the GLP1 increment response and reduced HOMA-%B, which might be potentially contributing to GLP1 resistance in patients with diabetes risk factors.KEYWORDS: diabetes risk, TCF7L2, GLP1, HOMA-%B

Mixed-Lineage Leukaemia Gene Regulates Glucose-Sensitive Gene Expression and Insulin Secretion in Pancreatic Beta Cells.

The escalating prevalence of diabetes mellitus underscores the need for a comprehensive understanding of pancreatic beta cell function. Interest in glucose effectiveness has prompted the exploration of novel regulatory factors. The myeloid/lymphoid or mixed-lineage leukaemia gene (MLL) is widely recognised for its role in leukemogenesis and nuclear regulatory mechanisms through its histone methyltransferase activity in active chromatin. However, its function within pancreatic endocrine tissues remains elusive. Herein, we unveil a novel role of MLL in glucose metabolism and insulin secretion. MLL knockdown in βHC-9 pancreatic beta cells diminished insulin secretion in response to glucose loading, paralleled by the downregulation of the glucose-sensitive genes SLC2a1 and SLC2a2. Similar observations were made in MLL heterozygous knockout mice (MLL+/-), which exhibited impaired glucose tolerance and reduced insulin secretion without morphological anomalies in pancreatic endocrine cells. The reduction in insulin secretion was independent of changes in beta cell mass or insulin granule morphology, suggesting the regulatory role of MLL in glucose-sensitive gene expression. The current results suggest that MLL interacts with circadian-related complexes to modulate the expression of glucose transporter genes, thereby regulating glucose sensing and insulin secretion. Our findings shed light on insulin secretion control, providing potential avenues for therapeutics against diabetes.

The liver-derived exosomes stimulate insulin gene expression in pancreatic beta cells under condition of insulin resistance.

An insufficient functional beta cell mass is a core pathological hallmark of type 2 diabetes (T2D). Despite the availability of several effective pharmaceuticals for diabetes management, there is an urgent need for novel medications to protect pancreatic beta cells under diabetic conditions. Integrative organ cross-communication controls the energy balance and glucose homeostasis. The liver and pancreatic islets have dynamic cross-communications where the liver can trigger a compensatory beta cell mass expansion and enhanced hormonal secretion in insulin-resistant conditions. However, the indispensable element(s) that foster beta cell proliferation and insulin secretion have yet to be completely identified. Exosomes are important extracellular vehicles (EVs) released by most cell types that transfer biological signal(s), including metabolic messengers such as miRNA and peptides, between cells and organs. We investigated whether beta cells can take up liver-derived exosomes and examined their impact on beta cell functional genes and insulin expression. Exosomes isolated from human liver HepG2 cells were characterized using various methods, including Transmission Electron Microscopy (TEM), dynamic light scattering (DLS), and Western blot analysis of exosomal markers. Exosome labeling and cell uptake were assessed using CM-Dil dye. The effect of liver cell-derived exosomes on Min6 beta cells was determined through gene expression analyses of beta cell markers and insulin using qPCR, as well as Akt signaling using Western blotting. Treatment of Min6 beta cells with exosomes isolated from human liver HepG2 cells treated with insulin receptor antagonist S961 significantly increased the expression of beta cell markers Pdx1, NeuroD1, and Ins1 compared to the exosomes isolated from untreated cells. In line with this, the activity of AKT kinase, an integral component of the insulin receptor pathway, is elevated in pancreatic beta cells, as represented by an increase in AKT's downstream substrate, FoxO1 phosphorylation. This study suggests that liver-derived exosomes may carry a specific molecular cargo that can affect insulin expression in pancreatic beta cells, ultimately affecting glucose homeostasis.

SAT045 Sexually Dimorphic Effects Of CCN5/WISP2 Overexpression On HFD-STZ Induced Diabetes And Glucose Intolerance

Abstract Disclosure: T. Alami: None. M. Zakikhani: None. V. Xega: None. J. Liu: None. Matricellular protein CCN5/WISP2 is a novel target of IGF-1 and WNT signaling within the pancreatic islets (1). Our previous works suggest that CCN5 promotes β-cell proliferation and survival via Akt/PKB and focal adhesion kinase. Last year, we reported sexually dimorphic effects of CCN5 knockout in glucose tolerance and insulin sensitivity in high-fat diet (HFD)-induced obesity, supporting a trophic role of endogenous CCN5 expression on pancreatic islets, albeit mostly in male mice (PSUN136, ENDO 2022). In order to further establish the in vivo effect of CCN5 in a gain-of-function approach, we assessed aP2-CCN5 transgenic mice which overexpress CCN5 in adipose tissues (2). These mice have increased serum level of CCN5, lean body mass and whole-body energy expenditure and hyperplastic adipose and cardiac tissues. Obese aP2-CCN5 mice remained insulin sensitive, glucose tolerant with a reduced insulin level. We first fed wild-type and transgenic mice, both male and female, a 60% HFD (Research Diets, D12492) for two weeks before administering 50 mg/kg streptozotocin (STZ) injections for 5 days, every other day. (i) In male mice, the HFD-STZ induced a steady elevation in glycemia from ∼9 to 25 mM in 7 weeks; CCN5 overexpression slowed down and curtailed the elevation to only 16 mM, and improved glucose tolerance at 48 days, suggesting a partial but significant protection from diabetes. (ii) Using the same dose of STZ, female wild-type mice exhibited only a slight elevation in glycemia from 8 to 12 mM. However, female aP2-CCN5 mice displayed a steady increase in glycemia to 18 mM and significantly impaired glucose tolerance at 16 and 48 days, suggesting rather worsening diabetes due to CCN5. In both male and female mice, there was no obvious difference in insulin sensitivity at 21 days nor in the overall weight loss caused by STZ injections. We are still analyzing the corresponding changes in serum insulin level and β-cell morphometry. Our results further support the notion that increased CCN5 level protects the β-cells, although only in male mice. The female environment, however, would abolish CCN5-induced protection. (1) Chowdhury S, Wang X, Srikant CB, Li Q, Fu M, Gong YJ, Ning G, Liu JL. IGF-I stimulates CCN5/WISP2 gene expression in pancreatic beta-cells, which promotes cell proliferation and survival against streptozotocin. Endocrinology. 2014; 155: 1629-1642. (2) Grunberg JR, Hoffmann JM, Hedjazifar S, Nerstedt A, Jenndahl L, Elvin J, Castellot J, Wei L, Moverare-Skrtic S, Ohlsson C, Holm LM, Backhed F, Syed I, Bosch F, Saghatelian A, Kahn BB, Hammarstedt A, Smith U. Overexpressing the novel autocrine/endocrine adipokine WISP2 induces hyperplasia of the heart, white and brown adipose tissues and prevents insulin resistance. Sci Rep. 2017; 7:43515 Presentation: Saturday, June 17, 2023

Beta cell dysfunction induced by bone morphogenetic protein (BMP)-2 is associated with histone modifications and decreased NeuroD1 chromatin binding

SummaryInsufficient insulin secretion is a hallmark of type 2 diabetes and has been attributed to beta cell identity loss characterized by decreased expression of several key beta cell genes. The pro-inflammatory factor BMP-2 is upregulated in islets of Langerhans from individuals with diabetes and acts as an inhibitor of beta cell function and proliferation. Exposure to BMP-2 induces expression of Id1-4, Hes-1, and Hey-1 which are transcriptional regulators associated with loss of differentiation. The aim of this study was to investigate the mechanism by which BMP-2 induces beta cell dysfunction and loss of cell maturity. Mouse islets exposed to BMP-2 for 10 days showed impaired glucose-stimulated insulin secretion and beta cell proliferation. BMP-2-induced beta cell dysfunction was associated with decreased expression of cell maturity and proliferation markers specific to the beta cell such as Ins1, Ucn3, and Ki67 and increased expression of Id1-4, Hes-1, and Hey-1. The top 30 most regulated proteins significantly correlated with corresponding mRNA expression. BMP-2-induced gene expression changes were associated with a predominant reduction in acetylation of H3K27 and a decrease in NeuroD1 chromatin binding activity. These results show that BMP-2 induces loss of beta cell maturity and suggest that remodeling of H3K27ac and decreased NeuroD1 DNA binding activity participate in the effect of BMP-2 on beta cell dysfunction.

349-OR: Selected DYRK1A Inhibitors Simultaneously Drive Both Human Beta-Cell Proliferation and Differentiation

Small molecule DYRK1A inhibitors induce adult human beta cell proliferation and expand human beta cell mass. Harmine also enhances differentiation and function in human beta cells in vitro and in vivo, increasing expression of key beta cell genes. It is unknown whether this “pro-differentiation effect” is a feature of all DYRK1A inhibitors. We compared harmine, 2-2c, 5-Iodotubericidin (5-IT), GNF4877, INDY, CC-401 and Leucettine-41 at doses that maximally drive human beta cell proliferation. As anticipated, harmine, 2-2c and 5-IT all increased expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC30A8, and ENTPD3. These effects were observed in normal human islets as well as in islets from donors with T2D. Surprisingly, in simultaneous experiments, the DYRK1A inhibitors GNF4877, INDY, CC-401 and Leucettine failed to induce these beta cell differentiation markers. The pro-differentiation effect of harmine was independent of DYRK1A inhibition: silencing DYRK1A in human islets induced beta cell proliferation, but had no effect on differentiation; harmine treatment in DYRK1A-silenced islets restored beta cell pro-differentiation effects. The pro-differentiation effect was also independent of NFAT signaling and DREAM Complex effects, since NFAT overexpression, or p130, p107, E2F4 and E2F5 silencing had no effect on beta cell differentiation. Similarly, silencing other known harmine targets (DYRK2, 3, 4, the CLK and HIPK family) had no effect on either human beta cell differentiation nor proliferation. In summary, harmine, 2-2c and 5-IT are unique among the DYRK1A inhibitor family in their simultaneous ability to enhance both beta cell proliferation and differentiation. While the beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor drug development. Disclosure P. Wang: None. K. Kumar: None. H. Liu: None. O. Wood: None. E. Karakose: None. L. Choleva: None. L. Lambertini: None. A. Garcia-Ocana: Consultant; Sun Pharmaceutical Industries Ltd. R.J. DeVita: None. A.F. Stewart: None.

1773-P: Glucocorticoid Receptor Signaling Regulates Pancreatic Beta-Cell Circadian Transcriptome and Function

Glucocorticoid (GC) signaling is an important regulator of glucose homeostasis. GC secretion also serves as an entrainment signal that coordinates circadian gene expression of peripheral circadian clocks. Pancreatic beta cells are key biological controllers of glucose homeostasis through the coordinated circadian release of insulin. However, the role of GC signaling in the regulation of beta cell circadian clocks and insulin secretion remains unknown. To address this, we first performed chromatin immunoprecipitation/sequencing (ChIP-seq) in beta cell lines (MIN6 and INS1) and identified >1,000 glucocorticoid receptor (GR)-bound regions (FDR<0.01) in response to the synthetic GC dexamethasone (DEX, 100nM). Annotation of GR-bound regions showed enrichment for pathways regulating insulin secretion and circadian rhythms. To further explore the effects of GC on circadian beta cell gene expression and function in vivo, we used C57BL/6 mice housed under either 12h/12h light-dark cycles (LD) or constant light-mediated circadian disruption (CD). Mice were subsequently administered daily intraperitoneal injections of DEX (2 mg/kg) or vehicle, designed to restore circadian secretion of GC disrupted in CD group. DEX treatment normalized circadian regulation of glucose tolerance (p<0.05 vs. vehicle) and glucose-stimulated insulin secretion (p<0.05 vs vehicle) in CD group. We next utilized RNA sequencing to assess circadian transcriptional profiles in islets isolated from LD, CD, and CD + DEX mice. Consistent with previous studies, CD resulted in abrogation of circadian islet mRNA expression, whereas DEX restored circadian rhythmicity and enhanced expression of core clock genes (e.g., Bmal1, Dbp, Nr1d1) and genes regulating beta cell identity (e.g., Ins1, Nkx6.1) and insulin secretion (e.g., Gck, Irs2). These data highlight the GC/GR axis as a direct regulator of beta cell circadian gene expression, insulin secretion and glucose tolerance. Disclosure S.K.Sen: None. L.Wang: None. M.Brown: None. J.Wu: None. Z.Wei: None. A.Matveyenko: None. Funding National Institutes of Health (DK098468)

Integrating genetics with single-cell multiomic measurements across disease states identifies mechanisms of beta cell dysfunction in type 2 diabetes.

Dysfunctional pancreatic islet beta cells are a hallmark of type 2 diabetes (T2D), but a comprehensive understanding of the underlying mechanisms, including gene dysregulation, is lacking. Here we integrate information from measurements of chromatin accessibility, gene expression and function in single beta cells with genetic association data to nominate disease-causal gene regulatory changes in T2D. Using machine learning on chromatin accessibility data from 34 nondiabetic, pre-T2D and T2D donors, we identify two transcriptionally and functionally distinct beta cell subtypes that undergo an abundance shift during T2D progression. Subtype-defining accessible chromatin is enriched for T2D risk variants, suggesting a causal contribution of subtype identity to T2D. Both beta cell subtypes exhibit activation of a stress-response transcriptional program and functional impairment in T2D, which is probably induced by the T2D-associated metabolic environment. Our findings demonstrate the power of multimodal single-cell measurements combined with machine learning for characterizing mechanisms of complex diseases.

Abstract C050: Elucidating mechanisms of endocrine-exocrine signaling in obesity-driven pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States. Various epidemiologic studies have shown that obesity increases the risk of PDAC. With rising rates of obesity worldwide, it is imperative that we understand the mechanisms by which obesity promotes PDAC progression. Therefore, to study the effect of obesity on PDAC, we combined a well-established genetic model of obesity (Lepob/ob) with a faithful oncogenic Kras-driven pancreatic cancer model (KC: Pdx1-Cre; KrasLSL-G12D/+) and showed that obese mice had both increased tumor burden and shortened survival compared to their lean counterparts. Strikingly, weight loss (via AAV-leptin rescue or caloric restriction), decreased tumor development, demonstrating that the tumor-promoting effects of obesity can be reversed. Bulk-RNA sequencing of tumors from obese mice exhibited aberrant expression of islet beta-cell genes, including those coding for the peptide hormones cholecystokinin (CCK) and amylin (Iapp). Single-cell RNA sequencing of islet beta-cells from wild-type, high fat diet-fed, and Lepob/ob mice revealed a trajectory of beta-cell adaptation to obesity with dysregulated hormone expression, including downregulation of insulin expression and upregulation of CCK and Iapp, concordant with loss of beta-cell identity transcription factors and beta-cell dysfunction. In silico analyses using dynamic optimal transport (TrajectoryNet) demonstrate that CCK-expressing beta-cells arise from pre-existing insulin low-expressing beta-cells, arguing for baseline beta-cell heterogeneity as a determinant of aberrant hormone expression. Lastly, transgenic expression of CCK or Iapp in beta-cells was sufficient to promote oncogenic Kras-driven pancreatic tumorigenesis, while converse beta-cell ablation impeded tumor progression in mice. Together, these results uncover a novel mechanism of obesity driven PDAC by local hormonal signaling between endocrine islets and exocrine acinar cells and reveal pancreatic endocrine adaptations that could be targeted to halt exocrine tumorigenesis. Citation Format: Cathy C. Garcia, Lauren Lawres, Sherry Agabiti, Jaffarguriqbal Singh, Alex Tong, Aarthi Venkat, Daniel B. Burkhardt, Rebecca Cardone, Richard G. Kibbey, Smita Krishnaswamy, Mandar D. Muzumdar. Elucidating mechanisms of endocrine-exocrine signaling in obesity-driven pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C050.

Proteasomal degradation of WT proinsulin in pancreatic beta cells

Preproinsulin entry into the endoplasmic reticulum yields proinsulin, and its subsequent delivery to the distal secretory pathway leads to processing, storage, and secretion of mature insulin. Multiple groups have reported that treatment of pancreatic beta cell lines, rodent pancreatic islets, or human islets with proteasome inhibitors leads to diminished proinsulin and insulin protein levels, diminished glucose-stimulated insulin secretion, and changes in beta-cell gene expression that ultimately lead to beta-cell death. However, these studies have mostly examined treatment times far beyond that needed to achieve acute proteasomal inhibition. Here, we report that although proteasomal inhibition immediately downregulates new proinsulin biosynthesis, it nevertheless acutely increases beta-cell proinsulin levels in pancreatic beta cell lines, rodent pancreatic islets, and human islets, indicating rescue of a pool of recently synthesized WT INS gene product that would otherwise be routed to proteasomal disposal. Our pharmacological evidence suggests that this disposal most likely reflects ongoing endoplasmic reticulum–associated protein degradation. However, we found that within 60 min after proteasomal inhibition, intracellular proinsulin levels begin to fall in conjunction with increased phosphorylation of eukaryotic initiation factor 2 alpha, which can be inhibited by blocking the general control nonderepressible 2 kinase. Together, these data demonstrate that a meaningful subfraction of newly synthesized INS gene product undergoes rapid proteasomal disposal. We propose that free amino acids derived from proteasomal proteolysis may potentially participate in suppressing general control nonderepressible 2 kinase activity to maintain ongoing proinsulin biosynthesis.

The Role of Fatty Acid Signaling in Islet Beta-Cell Adaptation to Normal Pregnancy

BackgroundMaintenance of a normal fetal nutrient supply requires major adaptations in maternal metabolic physiology, including of the islet beta-cell. The role of lipid signaling processes in the mechanisms of islet beta-cell adaptation to pregnancy has been minimally investigated.ObjectiveTo determine the effects of pregnancy on islet fatty acid (FA) metabolic partitioning and FA augmentation of glucose-stimulated insulin secretion (GSIS).MethodsAge matched virgin, early pregnant (gestational day-11, G11) and late pregnant (G19) Sprague-Dawley rats were studied. Fasted and fed state biochemistry, oral glucose tolerance tests (OGTT), and fasted and post-OGTT liver glycogen, were determined to assess in vivo metabolic characteristics. In isolated islets, FA (BSA-bound palmitate 0.25 mmol/l) augmentation of GSIS, FA partitioning into esterification and oxidation processes using metabolic tracer techniques, lipolysis by glycerol release, triacylglycerols (TG) content, and the expression of key beta-cell genes were determined.ResultsPlasma glucose in pregnancy was lower, including during the OGTT (glucose area under the curve 0-120 min (AUC0-120); 655±24 versus 849±13 mmol.l-1.min; G19 vs virgin; P<0.0001), with plasma insulin concentrations equivalent to those of virgin rats (insulin AUC0-120; 97±7 versus 83±7 ng.ml-1.min; G19 vs virgin; not significant). Liver glycogen was depleted in fasted G19 rats with full recovery after oral glucose. Serum TG increased during pregnancy (4.4±0.4, 6.7±0.5; 17.1±1.5 mmol/l; virgin, G11, G19, P<0.0001), and islet TG content decreased (147±42, 172±27, 73±13 ng/µg protein; virgin, G11, G19; P<0.01). GSIS in isolated islets was increased in G19 compared to virgin rats, and this effect was augmented in the presence of FA. FA esterification into phospholipids, monoacylglycerols and TG were increased, whereas FA oxidation was reduced, in islets of pregnant compared to virgin rats, with variable effects on lipolysis dependent on gestational age. Expression of Ppargc1a, a key regulator of mitochondrial metabolism, was reduced by 51% in G11 and 64% in G19 pregnant rat islets compared to virgin rat islets (P<0.001).ConclusionA lowered set-point for islet and hepatic glucose homeostasis in the pregnant rat has been confirmed. Islet adaptation to pregnancy includes increased FA esterification, reduced FA oxidation, and enhanced FA augmentation of glucose-stimulated insulin secretion.

Benefits of Sustained Upregulated Unimolecular GLP-1 and CCK Receptor Signalling in Obesity-Diabetes

Combined activation of GLP-1 and CCK1 receptors has potential to synergistically augment the appetite-suppressive and glucose homeostatic actions of the individual parent peptides. In the current study, pancreatic beta-cell benefits of combined GLP-1 and CCK1 receptor upregulation were established, before characterising bioactivity and antidiabetic efficacy of an acylated dual-acting GLP-1/CCK hybrid peptide, namely [Lys12Pal]Ex-4/CCK. Both exendin-4 and CCK exhibited (p<0.001) proliferative and anti-apoptotic effects in BRIN BD11 beta-cells. Proliferative benefits were significantly (p<0.01) augmented by combined peptide treatment when compared to either parent peptide alone. These effects were linked to increases (p<0.001) in GLUT2 and glucokinase beta-cell gene expression, with decreased (p<0.05-p<0.001) expression of NFκB and BAX. [Lys12Pal]Ex-4/CCK exhibited prominent insulinotropic actions in vitro, coupled with beneficial (p<0.001) satiety and glucose homeostatic effects in the mice, with bioactivity evident 24 h after administration. Following twice daily injection of [Lys12Pal]Ex-4/CCK for 28 days in diabetic high fat fed (HFF) mice with streptozotocin (STZ)-induced compromised beta-cells, there were clear reductions (p<0.05-p<0.001) in energy intake and body weight. Circulating glucose was returned to lean control concentrations, with associated increases (p<0.001) in plasma and pancreatic insulin levels. Glucose tolerance and insulin secretory responsiveness were significantly (p<0.05-p<0.001) improved by hybrid peptide therapy. In keeping with this, evaluation of pancreatic histology revealed restoration of normal islet alpha- to beta-cell ratios and reduction (p<0.01) in centralised islet glucagon staining. Improvements in pancreatic islet morphology were associated with increased (p<0.05) proliferation and reduced (p<0.001) apoptosis of beta-cells. Together, these data highlight the effectiveness of sustained dual GLP-1 and CCK1 receptor activation by [Lys12Pal]Ex-4/CCK for the treatment of obesity-related diabetes.

Efficient Restoration of Beta Cell Dedifferentiation by Calorie Restriction With High Fat/Low Carbohydrate Diet in Obese Diabetes Model and the Possible Role of GLP-1

In type 2 diabetes, pancreatic beta cells are gradually ‘exhausted’ and fall into beta cell dysfunction, which proceeds more severe insulin dependence. Among the proposed mechanisms of beta cell dysfunction such as endoplasmic reticulum stress and oxidative stress, the beta cell heterogeneity has attracted the researcher’s interest recently. In 2012, Talchai et al. revealed that the beta cells were dedifferentiated in diabetic mice model, and nowadays it is considered as one form of the beta cell heterogeneity and is observed broadly among diabetic animal models and human patients. Previously we showed that food restriction had the best effect to restore beta cell gene expression in obese diabetic model mice, among the known diabetic treatments which we tested. In the current study, we aimed to unveil the molecular basis in the improvement of beta cell dedifferentiation during the calorie restriction. First, we utilized the high-fat/low carbohydrate diet (HF) or low-fat/high carbohydrate (HC) diet, to determine whether fat restriction or sugar restriction reduces the beta cell dedifferentiation in obese mice. When calorie intake was restricted evenly, both HF diet and HC diet decreased the body weight and hyperglycemia in db/db mice equally. Albeit the same metabolic profile, db/db group fed with HC diet had more enlarged islets and more dedifferentiated beta cell features than db/dbs fed with HF diet, which indicated the compensatory beta cell response in HC diet group. Moreover, HC diet group showed more severe fatty liver than HF diet group, along with the elevated synthesis and accumulation of triglycerides and cholesterol in liver. It is speculated that the insulin resistance in liver might impact on the beta cell dedifferentiation. Next, we analyzed the effect of glucagon-like peptide 1 (GLP-1) on beta cell dedifferentiation, since GLP-1 is secreted more from intestine by protein and fat intake, rather than by sugar intake. Also, increasing number of reports have suggested the improving effect of GLP-1 on beta cell dysfunction and fatty liver. Indeed, GLP-1 administration altered the reduced beta cell/alpha cell ratio in db/db mice, which indicated the restoration of beta cell heterogeneity. We are now investigating if GLP-1 administration reimburse the beta cell dedifferentiation in db/db mice fed with HC diet, to illuminate the role of incretins in beta cell dedifferentiation induced by unbalanced nutrition during diet. Also, we will present the RNA sequencing data of the liver in db/db mice fed with HF and HC diet, to elucidate the key molecules and genes which connect the beta cell function and metabolic state in liver.

The Molecular Effect of Natural IAPP Variants on Pancreatic Beta Cell Gene Expression

Diabetes is the seventh leading cause of death and it is still likely to be underreported. Pancreatic beta cells, which are responsible for releasing insulin, have been found to undergo apoptosis in this disease, causing an imbalance in glucose reabsorption. Previous research has shown that islet amyloid polypeptide (IAPP), co-secreted with insulin, aggregates in these cells and contributes to their cell death. Therefore, finding inhibitors for this phenomenon is crucial to allow beta cells to live longer and increase survival rate and quality of life for patients with diabetes. We have found several natural IAPP variants that are inhibitors of human IAPP (hIAPP) aggregation. This work focuses on two natural IAPP variants – IAPPvR and IAPPvC.Both variants are capable of inhibiting human IAPP fiber formation and rescue the cells from the toxic effects of hIAPP. RNA seq results were used to identify differentially expressed genes responsible for the rescue. RNA seq data revealed several genes related to the unfolded protein response system and vesicle function. Further analysis is necessary to confirm the molecular mechanism responsible for this cellular response.

Imprinted Genes Impact Upon Beta Cell Function in the Current (and Potentially Next) Generation.

Beta cell failure lies at the centre of the aetiology and pathogenesis of type 2 diabetes and the epigenetic control of the expression of critical beta cell genes appears to play a major role in this decline. One such group of epigenetically-controlled genes, termed ‘imprinted’ genes, are characterised by transgenerational monoallelic expression due to differential allelic DNA methylation and play key functional roles within beta cells. Here, we review the evidence for this functional importance of imprinted genes in beta cells as well as their nutritional regulation by the diet and their altered methylation and/or expression in rodent models of diabetes and in type 2 diabetic islets. We also discuss imprinted genes in the context of the next generation, where dietary overnutrition in the parents can lead to their deregulation in the offspring, alongside beta cell dysfunction and defective glucose handling. Both the modulation of imprinted gene expression and the likelihood of developing type 2 diabetes in adulthood are susceptible to the impact of nutritional status in early life. Imprinted loci, therefore, represent an excellent opportunity with which to assess epigenomic changes in beta cells due to the diet in both the current and next generation.

Emerging Roles of Metallothioneins in Beta Cell Pathophysiology: Beyond and Above Metal Homeostasis and Antioxidant Response.

Simple SummaryDefective insulin secretion by pancreatic beta cells is key for the development of type 2 diabetes but the precise mechanisms involved are poorly understood. Metallothioneins are metal binding proteins whose precise biological roles have not been fully characterized. Available evidence indicated that Metallothioneins are protective cellular effectors involved in heavy metal detoxification, metal ion homeostasis and antioxidant defense. This concept has however been challenged by emerging evidence in different medical research fields revealing novel negative roles of Metallothioneins, including in the context of diabetes. In this review, we gather and analyze the available knowledge regarding the complex roles of Metallothioneins in pancreatic beta cell biology and insulin secretion. We comprehensively analyze the evidence showing positive effects of Metallothioneins on beta cell function and survival as well as the emerging evidence revealing negative effects and discuss the possible underlying mechanisms. We expose in parallel findings from other medical research fields and underscore unsettled questions. Then, we propose some future research directions to improve knowledge in the field.Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins whose precise biological roles have not been fully characterized. Existing evidence implicated MTs in heavy metal detoxification, metal ion homeostasis and antioxidant defense. MTs were thus categorized as protective effectors that contribute to cellular homeostasis and survival. This view has, however, been challenged by emerging evidence in different medical fields revealing novel pathophysiological roles of MTs, including inflammatory bowel disease, neurodegenerative disorders, carcinogenesis and diabetes. In the present focused review, we discuss the evidence for the role of MTs in pancreatic beta-cell biology and insulin secretion. We highlight the pattern of specific isoforms of MT gene expression in rodents and human beta-cells. We then discuss the mechanisms involved in the regulation of MTs in islets under physiological and pathological conditions, particularly type 2 diabetes, and analyze the evidence revealing adaptive and negative roles of MTs in beta-cells and the potential mechanisms involved. Finally, we underscore the unsettled questions in the field and propose some future research directions.

Pericytes contribute to the islet basement membranes to promote beta-cell gene expression

β-Cells depend on the islet basement membrane (BM). While some islet BM components are produced by endothelial cells (ECs), the source of others remains unknown. Pancreatic pericytes directly support β-cells through mostly unidentified secreted factors. Thus, we hypothesized that pericytes regulate β-cells through the production of BM components. Here, we show that pericytes produce multiple components of the mouse pancreatic and islet interstitial and BM matrices. Several of the pericyte-produced ECM components were previously implicated in β-cell physiology, including collagen IV, laminins, proteoglycans, fibronectin, nidogen, and hyaluronan. Compared to ECs, pancreatic pericytes produce significantly higher levels of α2 and α4 laminin chains, which constitute the peri-islet and vascular BM. We further found that the pericytic laminin isoforms differentially regulate mouse β-cells. Whereas α2 laminins promoted islet cell clustering, they did not affect gene expression. In contrast, culturing on Laminin-421 induced the expression of β-cell genes, including Ins1, MafA, and Glut2, and significantly improved glucose-stimulated insulin secretion. Thus, alongside ECs, pericytes are a significant source of the islet BM, which is essential for proper β-cell function.

Enhanced structure and function of human pluripotent stem cell-derived beta-cells cultured on extracellular matrix.

The differentiation of human stem cells into insulin secreting beta‐like cells holds great promise to treat diabetes. Current protocols drive stem cells through stages of directed differentiation and maturation and produce cells that secrete insulin in response to glucose. Further refinements are now needed to faithfully phenocopy the responses of normal beta cells. A critical factor in normal beta cell behavior is the islet microenvironment which plays a central role in beta cell survival, proliferation, gene expression and secretion. One important influence on native cell responses is the capillary basement membrane. In adult islets, each beta cell makes a point of contact with basement membrane protein secreted by vascular endothelial cells resulting in structural and functional polarization. Interaction with basement membrane proteins triggers local activation of focal adhesions, cell orientation, and targeting of insulin secretion. This study aims to identifying the role of basement membrane proteins on the structure and function of human embryonic stem cell and induced pluripotent stem cell‐derived beta cells. Here, we show that differentiated human stem cells‐derived spheroids do contain basement membrane proteins as a diffuse web‐like structure. However, the beta‐like cells within the spheroid do not polarize in response to this basement membrane. We demonstrate that 2D culture of the differentiated beta cells on to basement membrane proteins enforces cell polarity and favorably alters glucose dependent insulin secretion.

Preclinical evaluation of tyrosine kinase 2 inhibitors for human beta-cell protection in type 1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease leading to progressive loss of pancreatic beta cells. Interferon (IFN)-α plays a critical role in the crosstalk between pancreatic beta cells and the immune system in early insulitis. In human beta cells IFNα signals through JAK1 and TYK2, leading to endoplasmic reticulum stress, inflammation and HLA class I overexpression. IFNα, acting synergistically with IL-1β, induces apoptosis. Polymorphisms in TYK2 that decrease its activity are associated with protection against T1D, and we hypothesized that pharmacological inhibitors that specifically target TYK2 could protect human beta cells against the deleterious effects of IFNα. Two TYK2 inhibitors provided by Nimbus Lakshmi were tested in human insulin-producing EndoC-βH1 cells and human islets to evaluate their effect on IFNα signalling, beta-cell function and susceptibility to viral infection using RT-qPCR, western blot, immunofluorescence, ELISA and nuclear dyes. The two TYK2 inhibitors tested prevented IFNα-induced human beta-cell gene expression in a dose-dependent manner. They also protected human islets against IFNα + IL-1β-induced apoptosis. Importantly, these inhibitors did not modify beta-cell function or their survival following infection with the potential diabetogenic coxsackieviruses CVB1 and CVB5. The two TYK2 inhibitors tested inhibit the IFNα signalling pathway in human beta cells, decreasing its pro-inflammatory and pro-apoptotic effects without sensitizing the cells to viral infection. The preclinical findings could pave the way for future clinical trials with TYK2 inhibitors for the prevention and treatment of type 1 diabetes.

1875-P: The Impact of Carbohydrate/Fat Intake Balance on Beta-Cell Dedifferentiation and Fatty Liver in Obese Mouse Model

In clinical, the ’fatigue’ of the pancreatic beta cells is a famous phenomenon in type 2 diabetes. Nowadays, researchers describe the several mechanisms of beta cell dysfunction, such as glucolipotoxicity, endoplasmic reticulum stress and oxidative stress. In 2012, Talchai et al. revealed that the beta cells were dedifferentiated in diabetes, and previously we showed that food restriction had the most manifold good effects to restore beta cell gene expression, among the known diabetic treatments that we tested. In the current study, we aimed to unveil which nutrients should be cut down to protect beta cell differentiation, sugar or fat. First, we performed the pair-feeding with db/+ and db/db to reduce food intake of db/db mice. We utilized the high-fat/low carbohydrate diet (HF) or low-fat/high carbohydrate (HC) diet which were adjusted to the same total calorie intake. Pair fed-db/db mice had better body weight and glucose tolerance control than ad libitum db/db group, as previously we reported, and there was no significant difference between HF and HC diet. Albeit the same metabolic profile, HC diet group had more enlarged islets and more dedifferentiated beta cell features, which indicated the compensatory beta cell response in HC diet group. Moreover, HC diet group showed more severe fatty liver than HF diet group, along with the elevated synthesis and accumulation of triglycerides and cholesterol in liver. We are now investigating the relationship between sugar load, insulin resistance in liver and beta cell dedifferentiation. Disclosure E. Ishida: None. X. Lei: None. E. Yamada: None. S. Okada: None. M. Yamada: None. Funding Japan Society for the Promotion of Science (18K16222)