Β-cell Apoptosis Research Articles

Polysaccharide from Hovenia dulcis (Guaizao) improves pancreatic injury and regulates liver glycometabolism to alleviate STZ-induced type 1 diabetes mellitus in rats

Hovenia dulcis is a traditional medicinal and edible plant and has a major geographical presence in China. In this study, a polysaccharide purified from H. dulcis (HDPs-2A) was found to ameliorate type 1 diabetes mellitus (T1DM) in streptozotocin-induced diabetic rat. HDPs-2A treatment resulted in significantly lower fasting blood glucose levels, but higher body weight, plasma insulin, and liver glycogen levels. Moreover, HDPs-2A improved dyslipidemia, pancreatic oxidative stress, and reduced serum pro-inflammatory factors. In addition, HDPs-2A up-regulated PDX-1, activated and up-regulated IRS2 expression, and regulated apoptosis and regeneration of islet β cells to recover islet β-cell function injury in TIDM rats. HDPs-2A also up-regulated the expression of pancreatic GK and GLUT2 to improve insulin secretion ability of islet β-cells, ultimately improving the glucose metabolism disorder of T1DM rats. Moreover, HDPs-2A significantly up-regulated the expression of GK and down-regulated the expression of G6Pase in liver to improve liver glycogen synthesis, inhibit liver gluconiogenesis, and improve liver glucose metabolism disorder of T1DM rats. In summary, the hypoglycemic mechanisms of HDPs-2A may include regulating the regeneration and apoptosis of islet β-cells and activating liver glycometabolism-related signaling pathways in T1DM rats.

16-OR: Protein Kinase C d Mediates Cytokine-Induced ß-Cell Death via Upregulation of Pro-apoptotic Signaling

In type 1 diabetes (T1D) , autoreactive immune cells secrete cytokines, such as tumor necrosis factor-α (TNF-α) , interleukin-1β (IL-1β) , and interferon-γ (IFN-γ) that can initiate β-cell apoptosis. Protein kinase C delta (PKCδ) plays a role in mediating cytokine-induced β-cell apoptosis; however, the exact mechanisms and role in T1D are unknown. We hypothesize that PKCδ mediates cytokine-induced β-cell apoptosis via nuclear translocation and activation of pro-apoptotic signaling. Islets from WT or mice with a β-cell specific knockout of PKCδ (PKCδβ-KO) were cultured for 24hr with cytokines (10ng/ml TNFα, 5ng/ml IL-1β, 100ng/ml IFN-γ) , a caspase-3 inhibitor (Z-DEVD-FMK, 10µg/ml) , or an adenoviral vector for GFP-tagged PKCδ, GFP only, and a PKCδ mutant that cannot be cleaved by caspase-3 (CM-GFP-PKCδ) . Human islets were treated with 10μM PKCδ inhibitor (δV1-1) for 24hr with or without cytokines. Translocation was determined by GFP colocalization with a nuclear stain. Apoptosis was determined by fluorescent microscopy. PKCδ activity was determined with a FRET-based sensor in MIN6 cells. PKCδ’s role in activating pro-apoptotic signaling was determined via western blot. Decreasing PKCδ activity protected against cytokine-induced apoptosis in mouse (p=0.003) and human (p=0.020) islets. PKCδ translocation and activity increased in nuclei of cytokine treated islets (p<0.001, p=0.039) compared to untreated controls. Islets treated with caspase inhibitor (p<0.001) and CM-GFP-PKCδ islets were protected from cytokine-induced death. Western blot analysis demonstrates that PKCδ regulates pro-apoptotic signaling. Inhibiting PKCδ protected islets from cytokine-induced death. The role we identified for PKCδ in mediating pro-apoptotic proteins will provide novel insight to protect against cytokine-induced β-cell death. Our data suggests targeted inhibition of PKCδ has the potential to prevent cytokine-mediated β-cell death in T1D. Disclosure J.Collins: None. N.L.Farnsworth: None. R.K.Benninger: n/a. Funding American Diabetes Association (7-21-JDF-020) ; Juvenile Diabetes Research Foundation (1-FAC-2020-891-A-N)

14-OR: Harmine and Exenatide Combination Increases Human ß-Cell Survival

GLP1R agonists such as Exendin-4 (E) together with DYRK1A inhibitors such as Harmine (H) significantly increase human β-cell replication in vitro and in vivo. Importantly, 3-month treatment with H+E combination markedly increases human beta cell mass (∼7-fold) in islets transplanted in immunosuppressed mice, beyond the increase induced by proliferation. Therefore, we addressed whether H+E also enhances β-cell survival and that contributes to the increased human β-cell mass expansion in vivo. Treatment with H+E significantly decreased β-cell apoptosis in dispersed primary human islet cells treated in vitro with either thapsigargin (ER stress) , cytokines (inflammation) and H2O2 (ROS) , while the drugs alone or vehicle (V) did not induce such effect. Importantly, H+E treatment for 7 days significantly reduced β-cell apoptosis in human islet grafts transplanted in immunosuppressed mice assessed by insulin and TUNEL staining. Human β-cell mass analysis of these grafts by iDISCO+ revealed a significant ∼50% increase in H+E-treated mice compared with mice treated with drugs alone or V. Human α-cell mass was unchanged. To address the signaling pathways modulated by H+E after 7-day treatment, we performed RNA-seq analysis of these human islet grafts. We identified 29 differentially expressed human genes (> 2-fold, p<0.05) in H+E-treated human islet grafts. GSEA analysis revealed cell adhesion, survival, vascularization and secretion as the main pathways induced by H+E. Among the upregulated genes, VGF (VGF Nerve Growth Factor Inducible) is known to regulate insulin secretion and β-cell survival. We found that H+E treatment of human islets in vitro increases 2-to-3-fold VGF mRNA and secretion. We are now determining VGF involvement in H+E-induced human β-cell survival. In conclusion, we have uncovered a novel prosurvival function of H+E in human β-cells with the potential implication of VGF in these effects. These studies can lead to the discovery of future β-cell protection and regeneration therapies for diabetes. Disclosure C.Rosselot: None. A.F.Stewart: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Y.Li: None. D.Guevara: None. K.A.Beliard: None. R.Kang: None. P.Wang: None. K.Thakkar: None. G.Lu: None. R.J.Devita: None. Funding DYRK Inhibitors for Human Beta Cell Expansion RDK105015

10-OR: Combination Therapy with Anti-CD3 Antibody and Harmine plus Exendin-4 Enhances Remission from Recent-Onset Type 1 Diabetes.

Type 1 diabetes (T1D) results from loss of both immune tolerance and functional β-cells. Administration of harmine (H) plus exendin-4 (E) markedly induces human β-cell expansion. Anti-CD3 antibody treatment reduces C-peptide loss in T1D patients. Here, we tested whether combination therapy with anti-CD3 antibody and H+E enhances T1D remission in non-obese diabetic (NOD) mice. First, we tested whether H+E protects human β-cells against inflammation and ER stress. We found that H+E, but not the drugs alone, significantly reduced both thapsigargin- and cytokine-induced human β-cell apoptosis. Single-cell RNAseq of human islets treated with cytokines and H+E showed reduced IL1β and IFNγ signaling in β-cells. Apoptosis genes such as CYLD and RIPK1 were downregulated and prosurvival genes such as HIF1A and VEGFA were upregulated in β-cells of H+E-treated islets. Next, treatment of early-onset diabetic NOD mice with H+E (daily ip) for 8 weeks improved glucose homeostasis but failed to induce long-lasting immune tolerance and T1D remission. Therefore, we next treated early-onset diabetic NOD mice with low-dose anti-CD3 antibody daily for 3 days (40µg/day) followed by 8 weeks treatment with H+E or vehicle (V) (Alzet pumps) . Treatment with anti-CD3 and H+E eliminated hyperglycemia in 100% of the mice vs. only 20% remission in anti-CD3 and V-treated mice. CD3+ T cell numbers were significantly and similarly reduced (∼50%) in mice treated with H+E or V. Preliminary results indicate that activated T cells in pancreatic lymph nodes (PLNs) and islet insulitis were significantly reduced, and T regulatory cells in spleen and PLNs were increased in H+E-treated mice. TUNEL+ β-cells were decreased and Ki67+ β-cells were increased in the pancreas of H+E-treated mice. Collectively, these results indicate that combination therapy with low-dose anti-CD3 antibody and H+E enhances T1D remission in diabetic NOD mice by increasing β-cell viability and favoring immune tolerance. Disclosure G.Lu: None. R.Kang: None. Y.Li: None. P.Wang: None. C.Rosselot: None. R.J.Devita: None. A.F.Stewart: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Funding NIH DK105015

Quercetin protects against palmitate-induced pancreatic β-cell apoptosis by restoring lysosomal function and autophagic flux

Quercetin, a natural flavonoid, has been reported to prevent pancreatic β-cell apoptosis in animal models of diabetes. However, the underlying mechanism remains unclear. We investigated the mechanisms through which quercetin protects β cells from palmitate-induced apoptosis and determined whether autophagy is involved in this process. We found that quercetin treatment partially reduced palmitate-induced β-cell apoptosis. This protective effect was abolished by pharmacologic inhibition of autophagy and by silencing a key autophagy gene. Further analysis revealed that palmitate treatment promoted the expression of LC3 II, a marker of autophagosomes, but resulted in the blockade of autophagic flux due to lysosome dysfunction. Defective lysosome accumulation can cause lysosomal membrane permeabilization and the release of cathepsins from lysosome into the cytosol that triggers apoptosis. Treatment with quercetin reversed lysosomal dysfunction and promoted autophagosome-lysosome fusion, which restored defective autophagic flux and provoked autophagy. Overall, our results indicate that lysosomal dysfunction is a major factor that contributes to β-cell apoptosis and demonstrates that quercetin improves cell survival by restoring lysosomal function and autophagic flux. This study provides new evidence regarding the anti–apoptotic mechanism of quercetin in the treatment of type 2 diabetes.

Synthesis and Inhibitory Activity of Curculigoside A Derivatives as Potential Anti-Diabetic Agents with β-Cell Apoptosis

• It is the aim to synthesize aglycone of curculigoside A derivatives using acyl chloride in a mild condition by using imidazole and operating at room temperature. • Compounds 1, 2 , and 6 inhibit α-glucosidase are more effectively than acarbose. • Compounds 3, 4 , and 6 are inhibited TNF-α receptors better than or comparable to acarbose • Compound 6 has the potential to be an anti-diabetic by improving insulin resistance as well as inducing β-cell death. Diabetes mellitus (T1D, T2D, and DD) is a chronic metabolic condition that affected 4.2 million people in 2019 and is expected to reach 700 million by 2045. Hybrid or double diabetes (DD), which presents symptoms of both T1D and T2D, is caused by β-cell failure and apoptosis. The aglycone of curculigoside A derivatives was assessed in silico to optimize the pharmacokinetics and safety profile, as well as the binding mechanism and stability of anti-diabetic medicines with α-glucosidase. The objectives of this study are to synthesize, evaluate the biological activity of α-glucosidase, and simulate the inhibitory activity of the aglycone of curculigoside A derivatives on the tumor necrosis factor alpha (TNF-α) receptor as prospective anti-diabetic agents with β-cell apoptosis. Synthesis was carried out by using the esterification technique with halogenated carboxylic acids under moderate reaction conditions. Synthesized compounds 1 - 6 were assessed for their insulin resistance using α-glucosidase and investigated in silico by using the TNF-α receptor for its action in β-cell destruction. Compounds 3 - 6 have pharmacochemical properties that can be absorbed in the gastrointestinal tract, whereas compounds 1 and 2 are more water-soluble. The proposed methodology for the synthesis of the aglycone of curculigoside A derivatives by the esterification technique using halogenated carboxylic acids under moderate reaction conditions gives good yields. The biological evaluation study indicates that compounds 1, 2 , and 6 inhibit α-glucosidase more effectively than acarbose. By in silico study, compounds 3, 4 , and 6 inhibited TNF-α receptors are better than or comparable to acarbose, indicating that they have the ability to reduce β-cell apoptosis. In conclusion, 4-hydroxybenzyl-4-fluorobenzoat (compound 6 ) has the potential to be an anti-diabetic by inducing β-cell death.

Pancreatic INS-1 β-Cell Response to Thapsigargin and Rotenone: A Comparative Proteomics Analysis Uncovers Key Pathways of β-Cell Dysfunction.

Insulin-secreting β-cells in the pancreatic islets are exposed to various endogenous and exogenous stressing conditions, which may lead to β-cell dysfunction or apoptosis and ultimately to diabetes mellitus. However, the detailed molecular mechanisms underlying β-cell's inability to survive under severe stresses remain to be explored. This study used two common chemical stressors, thapsigargin and rotenone, to induce endoplasmic reticulum (ER) and mitochondria stress in a rat insuloma INS-1 832/13 β-cell line, mimicking the conditions experienced by dysfunctional β-cells. Proteomic changes of cells upon treatment with stressors at IC50 were profiled with TMT-based quantitative proteomics and further verified using label-free quantitive proteomics. The differentially expressed proteins under stress conditions were selected for in-depth bioinformatic analysis. Thapsigargin treatment specifically perturbed unfolded protein response (UPR) related pathways; in addition, 58 proteins not previously linked to the UPR related pathways were identified with consistent upregulation under stress induced by thapsigargin. Conversely, rotenone treatment resulted in significant proteome changes in key mitochondria regulatory pathways such as fatty acid β-oxidation, cellular respiration, citric acid cycle, and respiratory electron transport. Our data also demonstrated that both stressors increased reactive oxygen species production and depleted adenosine triphosphate synthesis, resulting in significant dysregulation of oxidative phosphorylation signaling pathways. These novel dysregulated proteins may suggest an alternative mechanism of action in β-cell dysfunction and provide potential targets for probing ER- and mitochondria stress-induced β-cell death.

Simulations of cross-amyloid aggregation of amyloid-β and islet amyloid polypeptide fragments

Amyloid-β (Aβ) and islet amyloid polypeptide (IAPP) are small peptides, classified as amyloids, that have the potential to self-assemble and form cytotoxic species, such as small soluble oligomers and large insoluble fibrils. The formation of Aβ aggregates facilitates the progression of Alzheimer’s disease (AD), while IAPP aggregates induce pancreatic β-cell apoptosis, leading to exacerbation of type 2 diabetes (T2D). Cross-amyloid interactions between Aβ and IAPP have been described both in vivo and in vitro, implying the role of Aβ or IAPP as modulators of cytotoxic self-aggregation of each species, and suggesting that Aβ-IAPP interactions are a potential molecular link between AD and T2D. Using molecular dynamics (MD) simulations, “hotspot” regions of the two peptides were studied to understand the formation of hexamers in a heterogeneous and homogeneous peptide-containing environment. Systems of only Aβ(16–22) peptides formed antiparallel, β-barrel-like structures, while systems of only IAPP(20–29) peptides formed stacked, parallel β-sheets and had relatively unstable aggregation structures after 2 μs of simulation time. Systems containing both Aβ and IAPP (1:1 ratio) hexamers showed antiparallel, β-barrel-like structures, with an interdigitated arrangement of Aβ(16–22) and IAPP(20–29). These β-barrel structures have features of cytotoxic amyloid species identified in previous literature. Ultimately, this work seeks to provide atomistic insight into both the mechanism behind cross-amyloid interactions and structural morphologies of these toxic amyloid species.

Functional Impact of Risk Gene Variants on the Autoimmune Responses in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that develops in the interplay between genetic and environmental factors. A majority of individuals who develop T1D have a HLA make up, that accounts for 50% of the genetic risk of disease. Besides these HLA haplotypes and the insulin region that importantly contribute to the heritable component, genome-wide association studies have identified many polymorphisms in over 60 non-HLA gene regions that also contribute to T1D susceptibility.Combining the risk genes in a score (T1D-GRS), significantly improved the prediction of disease progression in autoantibody positive individuals. Many of these minor-risk SNPs are associated with immune genes but how they influence the gene and protein expression and whether they cause functional changes on a cellular level remains a subject of investigation. A positive correlation between the genetic risk and the intensity of the peripheral autoimmune response was demonstrated both for HLA and non-HLA genetic risk variants. We also observed epigenetic and genetic modulation of several of these T1D susceptibility genes in dendritic cells (DCs) treated with vitamin D3 and dexamethasone to acquire tolerogenic properties as compared to immune activating DCs (mDC) illustrating the interaction between genes and environment that collectively determines risk for T1D. A notion that targeting such genes for therapeutic modulation could be compatible with correction of the impaired immune response, inspired us to review the current knowledge on the immune-related minor risk genes, their expression and function in immune cells, and how they may contribute to activation of autoreactive T cells, Treg function or β-cell apoptosis, thus contributing to development of the autoimmune disease.

Effects of Sodium-Glucose Co-Transporter-2 Inhibitors on Pancreatic β-Cell Mass and Function.

Sodium-glucose co-transporter-2 inhibitors (SGLT2is) not only have antihyperglycemic effects and are associated with a low risk of hypoglycemia but also have protective effects in organs, including the heart and kidneys. The pathophysiology of diabetes involves chronic hyperglycemia, which causes excessive demands on pancreatic β-cells, ultimately leading to decreases in β-cell mass and function. Because SGLT2is ameliorate hyperglycemia without acting directly on β-cells, they are thought to prevent β-cell failure by reducing glucose overload in this cell type. Several studies have shown that treatment with an SGLT2i increases β-cell proliferation and/or reduces β-cell apoptosis, resulting in the preservation of β-cell mass in animal models of diabetes. In addition, many clinical trials have shown that that SGLT2is improve β-cell function in individuals with type 2 diabetes. In this review, the preclinical and clinical data regarding the effects of SGLT2is on pancreatic β-cell mass and function are summarized and the protective effect of SGLT2is in β-cells is discussed.

Exosomal miR-140–3p and miR-143–3p from TGF-β1-treated pancreatic stellate cells target BCL2 mRNA to increase β-cell apoptosis

BackgroundPeople with chronic pancreatitis (CP) normally develop a fibrotic pancreas with reduced β-cell mass. Limited studies have focused on the development and pathogenesis of CP-related diabetes. MiRNAs packaged as exosomes are the key regulators of β-cell dysfunction. This study aimed to define the effect of exosomal miRNA from activated pancreatic stellate cells (PSCs) on β-cells. MethodsExosomes in the supernatants of mouse PSCs lines were extracted via ultracentrifugation and then identified. The role of exosomes secreted by transforming growth factor-β1 (TGF-β1)-treated PSCs in β-cell function was assessed. MiRNAs were prepared from exosomes extracted from TGF-β1-treated and untreated PSCs (T-Exo or C-Exo), and the miRNA expression profiles were compared by microarray. Then, miR-140–3p and miR-143–3p were overexpressed or inhibited in MIN6 cells and islets to determine their molecular and functional effects. ResultsExosomes were the predominant extracellular vesicles secreted by PSCs into the culture medium. The MIN6 cells incubated with T-Exo had less insulin secretion and lower viability than the MIN6 cells incubated with PBS or C-Exo. MiR-140–3p and miR-143–3p were notably upregulated in T-Exo. Enhancing the expression of miR-140–3p and miR-143–3p in β-cells decreased the cell count and viability and increased the cleaved caspase-3 levels. Mechanistically, T-Exo mediated the intercellular transfer of miR-140–3p and miR-143–3p by targeting the B-cell lymphoma 2 gene in recipient β-cells to induce cell death. ConclusionsExosomal miRNA transfer as a communication mode between PSCs and β-cells, which may be explored for its therapeutic utility.

Protection of pancreatic beta cells against high glucose-induced toxicity by astaxanthin-s-allyl cysteine diester: alteration of oxidative stress and apoptotic-related protein expression

Purpose: High glucose (HG)-induced oxidative stress is associated with apoptosis in pancreatic β-cells. The protective effect of astaxanthin-s-allyl cysteine diester (AST-SAC) against HG-induced oxidative stress in pancreatic β-cells (βTC-tet cell line) in in vitro was studied. Materials and Methods: βTC-tet cell line was exposed to HG in the presence and absence of AST-SAC. Various parameters such as cell viability, reactive oxygen species generation, mitochondrial membrane potential, DNA fragmentation and expression of proteins involved in apoptosis [p53, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax), cytochrome c and caspase 3] were studied. Results: Pre-treatment of βTC-tet cells with AST-SAC (4, 8 and 12 μg/ml) in the presence of HG (25 mM) protected the viability of the cells in a dose-dependent manner. AST-SAC treatment mitigated the oxidative stress thereby preventing the mitochondrial dysfunction, DNA damage and apoptosis in βTC-tet cells against HG toxicity. Treatment with AST-SAC prevented the increased expression of p53 under HG conditions. Further, AST-SAC treatment maintained the level of pro-apoptotic (Bax, cleaved caspase-3 and cytochrome c) and anti-apoptotic (Bcl-2) proteins to that of the control level under HG exposed conditions in βTC-tet cells. Conclusion: Altogether, AST-SAC alleviated HG-induced oxidative damage and apoptosis in pancreatic β-cells by enhancing the antioxidant status and altering apoptotic-related protein expression.

Differential effects of saturated and unsaturated free fatty acids on ferroptosis in rat β-cells

Elevated plasma concentrations of saturated free fatty acids (SFAs) are involved in pancreatic β-cell dysfunction and apoptosis, referred to as lipotoxicity. However, in contrast to apoptosis, the involvement of ferroptosis, as a distinct type of oxidative regulated cell death in β-cell lipotoxicity remains elusive. Therefore, the aim of this study was to determine the effects of various free fatty acids on ferroptosis induction in rat insulin-producing β-cells. Herein, rat insulin-producing β-cells underwent lipid peroxidation in the presence of long-chain SFAs and ω-6-polyunsaturated fatty acids (PUFAs), but only the latter induced ferroptosis. On the other hand, the ω-3-PUFA α-linolenate did not induce ferroptosis but sensitized insulin-producing β-cells to SFA-mediated lipid peroxidation. While the monounsaturated fatty acid oleate, overexpression of glutathione peroxidase 4, and the specific ferroptosis inhibitor ferrostatin-1 significantly abrogated lipid peroxidation, neither glutathione peroxidase 4 nor ferrostatin-1 affected palmitate-mediated toxicity. Site-specific expression of catalase in cytosol, mitochondria, and ER attenuated lipid peroxidation, indicating the contribution of metabolically generated H2O2 from all three subcellular compartments. These observations suggest that only ω-6-PUFAs reach the thresholds of lipid peroxidation required for ferroptosis, whereas SFAs favour apoptosis in β-cells. Hence, avoiding an excessive dietary intake of ω-6-PUFAs might be a crucial prerequisite for prevention of reactive oxygen species-mediated ferroptosis in insulin-producing cells.

Genetic Etiology of Neonatal Diabetes Mellitus in Vietnamese Infants and Characteristics of Those With INS Gene Mutations

BackgroundNeonatal diabetes mellitus (NDM) is a rare (1:90,000 newborns) but potentially devastating metabolic disorder characterized by hyperglycemia combined with low levels of insulin. Dominantly-acting insulin (INS) gene mutations cause permanent NDM through single amino acid changes in the protein sequence leading to protein misfolding, which is retained within the endoplasmic reticulum (ER), causing ER stress and β-cell apoptosis. Over 90 dominantly-acting INS gene mutations have been identified in individuals with permanent NDM.Patients and MethodsThe study included 70 infants diagnosed with NDM in the first year of life between May 2008 and May 2021 at the Vietnam National Children’s Hospital. Sequencing analysis of all the genes known to cause NDM was performed at the Exeter Genomic Laboratory, UK. Clinical characteristics, molecular genetics, and annual data relating to glycemic control (HbA1c) and severe hypoglycemia of those with INS mutations were collected. The main outcomes of interest were HbA1c, daily insulin dose, growth, and cognitive/motor development.ResultsFifty-five of 70 infants (78.5%) with NDM harbored a mutation in a known disease-causing gene and of these, 10 had six different de novo heterozygous INS mutations. Mean gestational age was 38.1 ± 2.5 weeks and mean birth weight was 2.8 ± 0.5 g. They presented with NDM at 20 ± 17 weeks of age; 6/10 had diabetic ketoacidosis with pH 7.13 ± 0.26; plasma glucose level 32.6 ± 14.3 mmol/l and HbA1C 81 ± 15% mmol/mol. After 5.5 ± 4.8 years of insulin treatment, 9/10 have normal development with a developmental quotient of 80-100% and HbA1C 64 ± 7.3 mmol/mol, 9/10 have normal height, weight, and BMI on follow-up.ConclusionsWe report a series of Vietnamese NDM cases with dominant INS mutations. INS mutations are the third commonest cause of permanent NDM. We recommend screening of the INS gene in all children diagnosed with diabetes in the first year of life.

Exenatide ameliorates hydrogen peroxide-induced pancreatic β-cell apoptosis through regulation of METTL3-mediated m6A methylation

Exenatide, a glucagon-like peptide-1 (GLP-1) receptor agonist, is a commonly used hypoglycemic agent in clinical practice; it inhibits reactive oxygen species-induced pancreatic β-cell apoptosis. N6-methyladenosine (m6A) is produced by the methylation of RNA N6 residues and has recently been shown to play a crucial role in the regulation of islet β-cell growth and development. However, the involvement of m6A methylation in the β-cell protective effects of exenatide has not been clarified. In this study, the m6A-methylated RNA content and methyltransferase-like 3 (METTL3) expression levels in NIT-1 cells and primary mouse islets were found to significantly decrease following treatment with hydrogen peroxide (H2O2). Treatment with exenatide induced an increase in m6A content and METTL3 expression in the H2O2-treated NIT-1 cells and islets. Moreover, METTL3 silencing resulted in NIT-1 cell apoptosis under normal culture conditions. METTL3 upregulation significantly ameliorated H2O2-induced apoptosis in NIT-1 cells and primary islets. Furthermore, the anti-apoptotic effects of exenatide were obviously reversed by METTL3 knockdown. In conclusion, these findings suggest that exenatide elicits its anti-apoptotic effects in pancreatic β-cells by promoting m6A methylation through the upregulation METTL3 expression.

Design, synthesis, and biological evaluation of a small molecule oral agonist of the glucagon-like-peptide-1 receptor

An absolute or relative deficiency of pancreatic β-cells mass and functionality is a crucial pathological feature common to type 1 diabetes mellitus and type 2 diabetes mellitus. Glucagon-like-peptide-1 receptor (GLP1R) agonists have been the focus of considerable research attention for their ability to protect β-cell mass and augment insulin secretion with no risk of hypoglycemia. Presently commercially available GLP1R agonists are peptides that limit their use due to cost, stability, and mode of administration. To address this drawback, strategically designed distinct sets of small molecules were docked on GLP1R ectodomain and compared with previously known small molecule GLP1R agonists. One of the small molecule PK2 (6-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxaline) displays stable binding with GLP1R ectodomain and induces GLP1R internalization and increasing cAMP levels. PK2 also increases insulin secretion in the INS-1 cells. The oral administration of PK2 protects against diabetes induced by multiple low-dose streptozotocin administration by lowering high blood glucose levels. Similar to GLP1R peptidic agonists, treatment of PK2 induces β-cell replication and attenuate β-cell apoptosis in STZ-treated mice. Mechanistically, this protection was associated with decreased thioredoxin-interacting protein expression, a potent inducer of diabetic β-cell apoptosis and dysfunction. Together, this report describes a small molecule, PK2, as an orally active nonpeptidic GLP1R agonist that has efficacy to preserve or restore functional β-cell mass.

IDF21-0041 Hyperglycemia aggravates apoptosis and neuroinflammation at the intermediate stage after post-traumatic brain injury

Background: Traumatic brain injury (TBI) is a highly lethal event with a poor prognosis. Recovering residual neuronal function in the intermediate stage of TBI is important for treatment; however, neuroinflammation and neuronal apoptosis impede residual neuronal repair processes. Hyperglycemia influences inflammatory processes and neuronal survival. Aim: We explored the effects of high glucose on neuroinflammation and neuronal death during the intermediate phase of TBI. Method: Rat models of type 2 diabetes mellitus and/or TBI were developed and behaviorally assessed. The histopathological changes were detected by Hematoxylin and eosin staining. Proinflammatory and anti-inflammatory in serum and hippocampal tissue were tested. And neural apoptosis was observed by Tunel staining and the expression of cleaved caspase-3 (CC-3) protein and Bax/Bcl-2 ratio were recorded. In in vitro assays, levels of inflammatory factors and apoptosis associated proteins were measured after MEK5 activation. Immunofluorescence colocalization of neuron-specific nuclear protein and p-MEK5 was analyzed by immunofluorescence cytochemistry. Results: Neurological function and cognitive abilities were impaired in TBI rats and worsened by type 2 diabetes mellitus. Histopathological staining and analyses of serum and hippocampal mRNA and protein levels indicated that neuroinflammation and apoptosis were induced in TBI rats and exacerbated by hyperglycemia. Hyperglycemia inhibited hippocampal MEK5 phosphorylation in TBI rats. In vitro assays were used to assess inflammatory factor expression, apoptotic protein levels and neuronal survival after MEK5 activation in TBI- and/or high-glucose-treated neurons. MEK5/extracellular signal-regulated kinase 5 (ERK5) pathway activation reduced the inflammation, cleaved caspase-3 expression, Bax/Bcl-2 ratio and apoptosis of TBI neurons, even under high-glucose conditions. Discussion: Our results indicated that HG exacerbated hippocampal injury and exaggerated the neurological deficits and learning/memory problems after TBI. We observed that HG aggravated neuroinflammation partly by inhibiting the MEK5/ERK5 signaling pathway. In addition to activating inflammation, HG has been reported to reduce neuronal survival by upregulating CC-3 and disrupting the balance between Bax and Bcl-2. The pro-apoptotic effects of HG have frequently been observed in individuals exposed to prolonged hyperglycemia. ERK5 was found to inhibit endoplasmic reticulum stress and apoptosis in pancreatic β-cells, thereby ameliorating the tissue damage caused by hyperglycemia. In the present study, activation of MEK5/ERK5 may reduced TBI cauded neuronal apoptosis. In conclusion, HG exacerbated hippocampal injury after TBI, largely by disrupting the homeostasis of pro- and anti-inflammatory cytokines and inducing hippocampal neuronal apoptosis. The MEK5/ERK5 pathway participated in both of these processes. These experimental findings emphasize the importance of good glycemic control for the functional recovery of residual neurons in TBI patients.

IDF21-0102 RANKL inhibitor Denosumab protects against glucolipotoxicity-induced β-cell dysfunction and apoptosis

Background: Denosumab is a human monoclonal antibody that binds to receptor activator of nuclear factor kappa-B ligand (RANKL). This inhibits the binding of RANKL to RANK on osteoclasts which reduces the activity of osteoclasts and avoids bone loss, and thus increases bone density. In addition, recent studies also showed that Denosumab seems to suppress blood sugar, but the underline mechanism is still unknown. Interestingly, some studies revealed the glucose-lowering effects by Denosumab may be related to the protection of β cells.

Effect of Metformin Intervention on Pancreatic β-Cell Apoptosis

Metformin is shown to have hypoglycemic effects. However, the relationship between metformin’s intervention in FFA-induced endoplasmic reticulum stress-mediated insulin resistance (IR) and insulin β-cell apoptosis under high-glucose condition remains unclear. Our study intends to assess their relationship. Human pancreatic β-cells were treated with metformin and cell proliferation and IR were detected by MTT assay along with detection of Wnt/β-catenin signaling by RT-PCR, cell cycle and apoptosis by flow cytometry. Metformin inhibited β cell proliferation which was mediated by FFA-induced endoplasmic reticulum stress in a time-dependent and dose-dependent manner as well as induced cell cycle arrest at G2/M phase. In addition, metformin inhibited β-catenin signaling activation and decreased the expression of c-myc, Dvl-2, survivin, Dvl-3, GSK-3β (p-ser9) and promoted GSK-3 (p-tyr216) and Axin-2 expression. In conclusion, metformin inhibits Wnt/β-catenin signaling and promotes FFA to induce endoplasmic reticulum stress, thereby mediating pancreatic β-cells behaviors.

A novel therapeutic combination of sitagliptin and melatonin regenerates pancreatic β-cells in mouse and human islets

Autoimmune-led challenge resulting in β-cell loss is responsible for the development of type 1 diabetes (T1D). Melatonin, a pineal hormone or sitagliptin, a dipeptidyl peptidase IV (DPP-IV) inhibitor, has increased β-cell mass in various diabetic models and has immunoregulatory property. Both β-cell regenerative capacity and melatonin secretion decrease with ageing. Thus, we aimed to investigate the therapeutic potential of melatonin combined with sitagliptin on β-cell regeneration under glucotoxic stress, in the streptozotocin-induced young and old diabetic mouse models, and euglycemic humanized islet transplant mouse model. Our results suggest that combination therapy of sitagliptin and melatonin show an additive effect in inducing mouse β-cell regeneration under glucotoxic stress, and in the human islet transplant mouse model. Further, in the young diabetic mouse model, the monotherapies induce β-cell transdifferentiation and reduce β-cell apoptosis whereas, in the old diabetic mouse model, melatonin and sitagliptin induce β-cell proliferation and β-cell transdifferentiation, and it also reduces β-cell apoptosis. Further, in both the models, combination therapy reduces fasting blood glucose levels, increases plasma insulin levels and glucose tolerance and promotes β-cell proliferation, β-cell transdifferentiation, and reduces β-cell apoptosis. It can be concluded that combination therapy is superior to monotherapies in ameliorating diabetic manifestations, and it can be used as a future therapy for β-cell regeneration in diabetes patients.