Beta-cell Death Research Articles

P29 Efecto de dieta con alto contenido en grasas saturadas, alcohol y un medio ambiente enriquecido en un modelo experimental de diabetes: estudio de alteraciones en marcadores metabólicos e histología de páncreas

Introducción: la diabetes mellitus tipo 2 (DM2) se caracteriza por ser una enfermedad inflamatoria, con afectación sistémica, crónica, plurimetabólica. Su mecanismo fisiopatogénico se caracteriza por glico-lipotoxicidad, estrés oxidativo crónico, que ocasionan hipoinsulinemia debido a la muerte de las células beta o su transdiferenciación.Objetivos: determinar el efecto de un ambiente enriquecido sobre marcadores metabólicos e histología pancreática en un modelo experimental de DM2.Materiales y métodos: se utilizaron 39 ratas Wistar macho de 12 meses edad al inicio del ensayo divididas en 5 grupos.Para alterar su metabolismo se utilizó una dieta con 30% de grasas saturadas (HFD) y/o consumo moderado (0,42 g/kg peso/día) de alcohol (Alc). El grupo con ambiente enriquecido (HFD+Alc+AE) se alojó en una jaula de mayor tamaño (250 dm3), con ruedas de correr y rampas, los otros animales en jaulas estándar de bioterio. El grupo control (C) se alimentó con dieta Chow. Se midieron marcadores metabólicos durante el ensayo de 16 meses de duración. Al finalizar el experimento se estudiaron histopatológicamente los páncreas de los animales. Los datos se analizaron mediante ANOVA y test t apareado (p=0,05).

Developmental Programming and Glucolipotoxicity: Insights on Beta Cell Inflammation and Diabetes.

Stimuli or insults during critical developmental transitions induce alterations in progeny anatomy, physiology, and metabolism that may be transient, sometimes reversible, but often durable, which defines programming. Glucolipotoxicity is the combined, synergistic, deleterious effect of simultaneously elevated glucose (chronic hyperglycemia) and saturated fatty acids (derived from high-fat diet overconsumption and subsequent metabolism) that are harmful to organs, micro-organs, and cells. Glucolipotoxicity induces beta cell death, dysfunction, and failure through endoplasmic reticulum and oxidative stress and inflammation. In beta cells, the misfolding of pro/insulin proteins beyond the cellular threshold triggers the unfolded protein response and endoplasmic reticulum stress. Consequentially there is incomplete and inadequate pro/insulin biosynthesis and impaired insulin secretion. Cellular stress triggers cellular inflammation, where immune cells migrate to, infiltrate, and amplify in beta cells, leading to beta cell inflammation. Endoplasmic reticulum stress reciprocally induces beta cell inflammation, whereas beta cell inflammation can self-activate and further exacerbate its inflammation. These metabolic sequelae reflect the vicious cycle of beta cell stress and inflammation in the pathophysiology of diabetes.

Modulation of Rab7a-mediated growth factor receptor trafficking inhibits islet beta cell apoptosis and autophagy under conditions of metabolic stress

Regenerative medicine approaches to enhancing beta cell growth and survival represent potential treatments for diabetes. It is known that growth factors such as insulin, IGF-1 and HGF support beta cell growth and survival, but in people with type 2 diabetes the destructive effects of metabolic stress predominate and beta cell death or dysfunction occurs. In this study we explore the novel hypothesis that regulation of growth factor receptor trafficking can be used to promote islet beta cell survival. Growth factor signalling is dependent on the presence of cell surface receptors. Endosomal trafficking and subsequent recycling or degradation of these receptors is controlled by the Rab GTPase family of proteins. We show that Rab7a siRNA inhibition enhances IGF-1 and HGF signalling in beta cells and increases expression of the growth factor receptors IGF-1R and c-Met. Furthermore, Rab7a inhibition promotes beta cell growth and islet survival, and protects against activation of apoptosis and autophagy pathways under conditions of metabolic stress. This study therefore demonstrates that Rab7a-mediated trafficking of growth factor receptors controls beta cell survival. Pharmaceutical Rab7a inhibition may provide a means to promote beta cell survival in the context of metabolic stress and prevent the onset of type 2 diabetes.

Inhibition of heparanase protects against pancreatic beta cell death in streptozotocin-induced diabetic mice via reducing intra-islet inflammatory cell infiltration.

Intra-islet heparan sulfate (HS) plays an important role in the maintenance of pancreatic islet function. The aim of this study was to investigate the effect mechanism of HS loss on the functioning of islets in diabetic mice. The hypoglycaemic effect of a heparanase inhibitor, OGT2115, was tested in a streptozotocin-induced diabetic mouse model. The islets in pancreatic sections were also stained to reveal their morphology. An insulinoma cell line (MIN6) and primary isolated murine islets were used to investigate the effect of OGT2115 in vitro. Intra-islet HS was clearly lost in streptozotocin-induced diabetic mice due to the increased heparanase expression in damaged islets. OGT2115 prevented intra-islet HS loss and improved the glucose profile and insulin secretion in streptozotocin-treated mice. The apoptosis of pancreatic beta cells and the infiltration of mononuclear macrophages, CD4- and CD8-positive T-cells in islets was reduced by OGT2115 in streptozotocin-treated mice, but OGT2115 did not alter the direct streptozotocin-induced damage in vitro. The expression of heparanase was increased in high glucose-treated isolated islets but not in response to direct streptozotocin stimulation. Further experiments showed that high glucose stimuli could decreased expression of PPARγ in cultured islets, thereby relieving the PPARγ-induced inhibition of heparanase gene expression. Hyperglycaemia could cause intra-islet HS loss by elevating the expression of heparanase, thereby aggravating inflammatory cell infiltration and islet damage. Inhibition of heparanase might provide benefit for pancreatic beta cell protection in Type 1 diabetes.

ATM and P53 differentially regulate pancreatic beta cell survival in Ins1E cells.

Pancreatic beta cell death is a hallmark of type 1 and 2 diabetes (T1D/T2D), but the underlying molecular mechanisms are incompletely understood. Key proteins of the DNA damage response (DDR), including tumor protein P53 (P53, also known as TP53 or TRP53 in rodents) and Ataxia Telangiectasia Mutated (ATM), a kinase known to act upstream of P53, have been associated with T2D. Here we test and compare the effect of ATM and P53 ablation on beta cell survival in the rat beta cell line Ins1E. We demonstrate that ATM and P53 differentially regulate beta cell apoptosis induced upon fundamentally different types of diabetogenic beta cell stress, including DNA damage, inflammation, lipotoxicity and endoplasmic reticulum (ER) stress. DNA damage induced apoptosis by treatment with the commonly used diabetogenic agent streptozotocin (STZ) is regulated by both ATM and P53. We show that ATM is a key STZ induced activator of P53 and that amelioration of STZ induced cell death by inhibition of ATM mainly depends on P53. While both P53 and ATM control lipotoxic beta cell apoptosis, ATM but not P53 fails to alter inflammatory beta cell death. In contrast, tunicamycin induced (ER stress associated) apoptosis is further increased by ATM knockdown or inhibition, but not by P53 knockdown. Our results reveal differential roles for P53 and ATM in beta cell survival in vitro in the context of four key pathophysiological types of diabetogenic beta cell stress, and indicate that ATM can use P53 independent signaling pathways to modify beta cell survival, dependent on the cellular insult.

Low concentration IL-1β promotes islet amyloid formation by increasing hIAPP release from humanised mouse islets in vitro.

Aggregation of the beta cell secretory product human islet amyloid polypeptide (hIAPP) results in islet amyloid deposition, a pathological feature of type 2 diabetes. Amyloid formation is associated with increased levels of islet IL-1β as well as beta cell dysfunction and death, but the mechanisms that promote amyloid deposition in situ remain unclear. We hypothesised that physiologically relevant concentrations of IL-1β stimulate beta cell islet amyloid polypeptide (IAPP) release and promote amyloid formation. We used a humanised mouse model of endogenous beta cell hIAPP expression to examine whether low (pg/ml) concentrations of IL-1β promote islet amyloid formation in vitro. Amyloid-forming islets were cultured for 48h in the presence or absence of IL-1β with or without an IL-1β neutralising antibody. Islet morphology was assessed by immunohistochemistry and islet mRNA expression, hormone content and release were also quantified. Cell-free thioflavin T assays were used to monitor hIAPP aggregation kinetics in the presence and absence of IL-1β. Treatment with a low concentration of IL-1β (4pg/ml) for 48h increased islet amyloid prevalence (93.52 ± 3.89% vs 43.83 ± 9.67% amyloid-containing islets) and amyloid severity (4.45 ± 0.82% vs 2.16 ± 0.50% amyloid area/islet area) in hIAPP-expressing mouse islets in vitro. This effect of IL-1β was reduced when hIAPP-expressing islets were co-treated with an IL-1β neutralising antibody. Cell-free hIAPP aggregation assays showed no effect of IL-1β on hIAPP aggregation in vitro. Low concentration IL-1β did not increase markers of the unfolded protein response (Atf4, Ddit3) or alter proIAPP processing enzyme gene expression (Pcsk1, Pcsk2, Cpe) in hIAPP-expressing islets. However, release of IAPP and insulin were increased over 48h in IL-1β-treated vs control islets (IAPP 0.409 ± 0.082 vs 0.165 ± 0.051pmol/5 islets; insulin 87.5 ± 8.81 vs 48.3 ± 17.3pmol/5 islets), and this effect was blocked by co-treatment with IL-1β neutralising antibody. Under amyloidogenic conditions, physiologically relevant levels of IL-1β promote islet amyloid formation by increasing beta cell release of IAPP. Neutralisation of this effect of IL-1β may decrease the deleterious effects of islet amyloid formation on beta cell function and survival.

Targeting polyamine biosynthesis to stimulate beta cell regeneration in zebrafish

ABSTRACT Type 1 diabetes (T1D) is a disease characterized by destruction of the insulin-producing beta cells. Currently, there remains a critical gap in our understanding of how to reverse or prevent beta cell loss in individuals with T1D. Previous studies in mice discovered that pharmacologically inhibiting polyamine biosynthesis using difluoromethylornithine (DFMO) resulted in preserved beta cell function and mass. Similarly, treatment of non-obese diabetic mice with the tyrosine kinase inhibitor Imatinib mesylate reversed diabetes. The promising findings from these animal studies resulted in the initiation of two separate clinical trials that would repurpose either DFMO (NCT02384889) or Imatinib (NCT01781975) and determine effects on diabetes outcomes; however, whether these drugs directly stimulated beta cell growth remained unknown. To address this, we used the zebrafish model system to determine pharmacological impact on beta cell regeneration. After induction of beta cell death, zebrafish embryos were treated with either DFMO or Imatinib. Neither drug altered whole-body growth or exocrine pancreas length. Embryos treated with Imatinib showed no effect on beta cell regeneration; however, excitingly, DFMO enhanced beta cell regeneration. These data suggest that pharmacological inhibition of polyamine biosynthesis may be a promising therapeutic option to stimulate beta cell regeneration in the setting of diabetes.

Loss of Caveolin-1 Is Associated with a Decrease in Beta Cell Death in Mice on a High Fat Diet.

Elevated free fatty acids (FFAs) impair beta cell function and reduce beta cell mass as a consequence of the lipotoxicity that occurs in type 2 diabetes (T2D). We previously reported that the membrane protein caveolin-1 (CAV1) sensitizes to palmitate-induced apoptosis in the beta pancreatic cell line MIN6. Thus, our hypothesis was that CAV1 knock-out (CAV1 KO) mice subjected to a high fat diet (HFD) should suffer less damage to beta cells than wild type (WT) mice. Here, we evaluated the in vivo response of beta cells in the pancreatic islets of 8-week-old C57Bl/6J CAV1 KO mice subjected to a control diet (CD, 14% kcal fat) or a HFD (60% kcal fat) for 12 weeks. We observed that CAV1 KO mice were resistant to weight gain when on HFD, although they had high serum cholesterol and FFA levels, impaired glucose tolerance and were insulin resistant. Some of these alterations were also observed in mice on CD. Interestingly, KO mice fed with HFD showed an adaptive response of the pancreatic beta cells and exhibited a significant decrease in beta cell apoptosis in their islets compared to WT mice. These in vivo results suggest that although the CAV1 KO mice are metabolically unhealthy, they adapt better to a HFD than WT mice. To shed light on the possible signaling pathway(s) involved, MIN6 murine beta cells expressing (MIN6 CAV) or not expressing (MIN6 Mock) CAV1 were incubated with the saturated fatty acid palmitate in the presence of mitogen-activated protein kinase inhibitors. Western blot analysis revealed that CAV1 enhanced palmitate-induced JNK, p38 and ERK phosphorylation in MIN6 CAV1 cells. Moreover, all the MAPK inhibitors partially restored MIN6 viability, but the effect was most notable with the ERK inhibitor. In conclusion, our results suggest that CAV1 KO mice adapted better to a HFD despite their altered metabolic state and that this may at least in part be due to reduced beta cell damage. Moreover, they indicate that the ability of CAV1 to increase sensitivity to FFAs may be mediated by MAPK and particularly ERK activation.

Late Pathomorphological Features of the Endocrine Pancreas in Patients With Type 2 Diabetes Mellitus.

IntroductionIslet amyloid polypeptide (IAPP) amyloidosis is a pathologic alteration of the pancreas, represented by abnormal accumulation of amylin in the interstitial tissue. Amylin is a neuroendocrine hormone, co-secreted with insulin by beta cells and participating in downstream regulation of postprandial glycemia. This report aims to examine IAPP amyloidosis as a late consequence of poor control of blood glucose levels in patients with type 2 diabetes mellitus (T2DM) who have been referred for autopsy.Materials and methodsA total of 34 consecutive autopsies performed at the St. Marina University Hospital, Varna, Bulgaria, carried out by a single pathologist were included in the study. Samples from the tail of the pancreas were obtained to evaluate the state of the changes and were analyzed together with the specific organ changes associated with T2DM, as well as the medical documentation of the patients.ResultsOf the 34 autopsies, 10 cases (six females and four males) were included in the study, seven of whom had a medical history of T2D. The average age was 65.7 years (range 50 to 85 years). In all of the cases, morphological features of fibrosis and lipomatosis were present, with one of the patients having signs of pancreatic amyloidosis - Congo red positive deposition of pink, amorphous material in the extracellular matrix.ConclusionThe described pathological alterations in all of the cases illustrate the progressing impairment of the structure of the pancreas, especially beta cells dysfunction in late stages of T2D, and highlight IAPP amyloidosis as the cause of irreversible damage of the isles of Langerhans and beta cell death.

79-OR: Signal-Dependent Chromatin Accessibility Dynamics in Pancreatic Islet Dysfunction

In type 2 diabetes (T2D), inflammation induces massive changes in beta cell transcriptome and epigenome, resulting in eventual dysfunction and death of beta cells. Accordingly, strategies designed to protect beta cells from inflammatory stress at transcriptional and epigenetic level could be attractive for combating diabetes. Through genome-wide CRISPR screening in human stem cell differentiated islet like organoids, we identified a novel mechanism connecting signal- dependent transcription by vitamin D receptor (VDR) and fine-tuning of chromatin accessibility by a balance between two SWI/SNF complexes, BAF and PBAF. Our studies revealed that VDR recruits BAF and PBAF complexes through two bromodomain proteins, BRD9 and BRD7, respectively. The balance between BAF-BRD9 and PBAF-BRD7 determines the VDR-driven anti-inflammatory and pro-survival response in pancreatic beta cells. In a dual regulatory mechanism, inhibition of the VDR-BRD9 interaction in combination with ligand activation of VDR cooperate to dismiss the BAF-BRD9 complex and shift the balance to the activating PBAF-BRD7 complex to induce a coordinated transcriptional response. Notably, pharmacologically potentiated VDR signaling by a synthetic ligand in combination with a BRD9 inhibitor can partially restore beta cell function and glucose homeostasis in various T2D mouse models. Tissue specific models further confirmed the functional role of VDR and BRD9 in beta cell stress response in vivo. Together, our results revealed that an unexpected epigenetic balance between the bromodomain readers has a major impact on beta cell survival and glucose homeostasis, and demonstrated the therapeutic potential of targeting inflammatory/metabolic stress through synergistic modulation of hormone receptors and chromatin accessibility. Disclosure Z. Wei: None. Funding National Institutes of Health (1K01DK120808)

51-OR: Loss of Critical ER Chaperone GRP78 Leads to Beta-Cell Death by a JNK Dependent Mechanism

Endoplasmic reticulum (ER) health is an important determinant of pancreatic beta cell function or failure. Adaptive ER stress leads to expansion of beta cell number through proliferation, but decompensated ER stress results in beta cell loss and contributes to both T1D and T2D. Therefore, understanding the mechanisms of ER-stress-induced beta cell death could help identify novel strategies for the prevention or treatment of diabetes. Glucose responsive protein GRP78/HSPA5 is a key ER chaperone. Loss of GRP78 causes cell death in many cell types; including, in our own prior work, the beta cell. To study the mechanism by which GRP78 loss leads to beta cell death we devised an ex vivo model of GRP78 knockdown by transducing Grp78-flox mouse islets with adenovirus expressing Cre recombinase. To study human islets, we used adenovirus expressing shRNA against GRP78. Using these tools, we successfully reduced GRP78 RNA and protein in primary mouse and human islet cells. Loss of GRP78 strongly activated the ATF6, IRE1 and PERK pathways of the unfolded protein response (UPR). GRP78 knockdown decreased beta cell number due to increased cell death as measured by Annexin V and TUNEL staining. Probing the mechanisms of cell loss, we found that loss of GRP78 activated multiple death-related pathways, notably autophagy and apoptosis; cell death effectors Chop, caspase 9 and cleaved caspase 3 were increased. Additionally, GRP78 deletion increased stress kinase JNK (c-Jun-N-terminal kinase) which has been implicated in cellular proliferation as well as cell death. Inhibition of JNK kinase using JNK-IN-8 inhibitor during GRP78 loss conditions decreased phosphorylation of JNK and rescued beta cells from cell death as measured with Annexin V and TUNEL. Therefore, we conclude that ER stress related cell death induced by loss of GRP78 (ER stress) involves JNK activation, and that modulation of JNK under certain circumstances may help maintain beta cell mass to protect against diabetes. Disclosure R.B. Sharma: None. C.O. Darko: None. B. Gablaski: None. A.S. Lee: None. L.C. Alonso: Consultant; Self; Fairbanks Pharmaceuticals Inc. Research Support; Self; American Diabetes Association. Funding National Institute of Diabetes and Digestive and Kidney Diseases (5R01DK113300-02)

86-OR: High-Fat Diet Prevents Autoimmune Diabetes in NOD Mice

Type 1 diabetes (T1D) has a strong genetic predisposition and requires an environmental trigger to initiate autoimmune destruction of beta cells. The rate of childhood obesity has risen in parallel to the rate of T1D, suggesting high fat diet (HFD) may be an environmental trigger in the development of autoimmune diabetes. Supporting this hypothesis, HFD can induce insulin resistance, beta cell stress, dysfunctional insulin production, and beta cell death. To explore the role of HFD in the development of T1D, 4 week-old non-obese diabetic (NOD) female mice were placed on HFD or chow diet (CD). At 10 weeks of age NOD mice fed with HFD demonstrated a significant increase in body weight and non-fasting blood glucose along with mild glucose intolerance compared with NOD CD fed mice. Histological analysis at 10 weeks of age revealed significantly less pancreatic immune cell infiltration and increased beta cell mass in HFD fed mice compared to CD fed mice. By 15 weeks of age, HFD fed NOD animals demonstrated impaired glucose tolerance similar to CD fed mice, increased glucose-stimulated insulin secretion and total insulin content, and insulin resistance. Surprisingly, at 25 weeks of age the HFD fed NOD mice were completely protected from the development of diabetes, while as expected 70% of NOD CD fed mice had developed diabetes. In summary, this study demonstrates that in the NOD model of T1D HFD reduced islet immune cell infiltration, increased beta cell mass, insulin content and insulin secretion protecting the mice from development of diabetes. The mechanism underlying this protection seems to be two-fold: expansion of beta cell mass and prevention of autoimmune-mediated beta cell death. Our data correlate with other recent studies indicating that early stimulation of beta cell replication can induce immune tolerance in NOD animals preventing T1D. Identification of the mechanisms by which HFD leads to prevention of diabetes in NOD animals may lead to the development of novel therapeutic interventions to prevent T1D in humans. Disclosure A.L. Clark: None. Z. Yan: None. S.X. Chen: None. M. Fuess: None. G. McGinn: None. M.S. Remedi: None. Funding National Institutes of Health (P30DK020579, K12HD076224-04)

2115-P: Beta-Cell Autophagy Is Reduced in Type 1 Diabetes

Autophagy is a dynamic recycling mechanism that maintains beta cell homeostasis, whereas the related process of crinophagy also plays a specific role in insulin granule regulation. Impairment of autophagy and crinophagy have been implicated in beta cell death and dysfunction in the context of type 2 diabetes; however, there is currently no literature describing beta cell autophagy in type 1 diabetes pathogenesis. Therefore, we set out to determine if beta cell autophagy and crinophagy are impaired in the context of type 1 diabetes. We utilized immunofluorescent staining of LC3 (autophagosome marker), Lamp1 (lysosome marker), and proinsulin to analyze pancreatic islet autophagy and crinophagy in the NOD (non-obese diabetic) mouse model of type 1 diabetes as well as in human pancreas tissue obtained from the network for Pancreatic Organ donors with Diabetes (nPOD). We observed evidence of reduced macroautophagy in the residual proinsulin-positive islet cells of human donors with type 1 diabetescompared to controls, as measured by significantly reduced autophagosome:lysosome colocalization (p=0.034). Similarly, there was a trend towards reduced autophagosome:lysosome colocalization in islets of diabetic NOD mice compared to nondiabetic NOR mice (p=0.08). We also observed significantly reduced colocalization of proinsulin with lysosomes in the proinsulin positive cells from human donors with type 1 diabetes in comparison to both controls without diabetes (p=0.0014) and autoantibody positive donors (p= 0.0278), as well as in diabetic NOD vs. nondiabetic NOR mice (p=0.0121), suggesting that crinophagy is also reduced as a function of type 1 diabetes. Collectively, we provide evidence that macroautophagy and crinophagy are reduced in the context of type 1 diabetes. Considering these data in the context of what is known regarding the cell-protective effects of islet autophagy, we suggest targeting beta cell autophagy pathways as an approach to reduce apoptosis in individuals at risk for type 1 diabetes development. Disclosure C. Muralidharan: None. M. Marasco: Employee; Spouse/Partner; Eli Lilly and Company. J. Crowder: Employee; Spouse/Partner; Eli Lilly and Company. A.M. Conteh: None. A.K. Linnemann: None.

50-OR: Beta Cell GDF15 Contributes to Diabetic Stress-Induced Beta-Cell Loss

Endoplasmic reticulum (ER) stress has been shown to play an important role in beta cell loss in diabetes. However, the factors involved in this process are still not fully understood. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor beta (TGFβ) family, is a stress response gene and is involved in various diseases. GDF15 is expressed in adult human beta cells; however, its role in beta cells and diabetes had not been elucidated. Interestingly, we now have identified GDF15 as a key factor involved in ER stress-induced beta cell loss. We found that GDF15 mRNA, protein levels and promoter activity were significantly induced by ER stress in INS-1 cells, Akita beta cells, and primary human islets and we identified the transcription factor involved. Moreover, we discovered that overexpression of GDF15 exacerbated, while knockdown of GDF15 inhibited ER stress-induced beta cell apoptosis, at least in part by regulating the pro-apoptotic factor CHOP. Most importantly, we found that GDF15 knockout mice were protected from STZ-induced diabetes, maintained insulin producing beta cells and exhibited improved serum insulin levels. Taken together, our findings suggest that GDF15 plays an important role in ER stress-induced beta cell death and that inhibition of GDF15 may represent a novel strategy to reduce beta cell loss and treat diabetes. Disclosure G. Xu: None. S. Jo: None. J. Chen: None. T. Grayson: None. A. Shalev: None.

84-OR: Laminin-Islet Interactions Protect against Cytokine-Mediated Beta-Cell Death via Down-Regulation of Protein Kinase C Delta

Islets are surrounded by a specialized extracellular matrix (ECM). Interactions with the peri-islet ECM, specifically laminin, promote islet viability and function. In T1D during immune infiltration the peri-islet ECM is broken down and β-cell death occurs in part through production of high levels of Th1 cytokines. Recent work has shown that cytokines alter islet function via protein kinase C Δ (PKCΔ) dependent mechanisms and that islet interactions with laminin reduce PKCΔ activity. We hypothesize that laminin-islet interactions protect against cytokine-mediated β-cell death via downregulation of PKCΔ. To test our hypothesis, we used a 3D reverse thermal gel (RTG) scaffold with either laminin-10 (RTG-Lam) or lysine (RTG-Lys) as a control. Islets were isolated from WT or mice with an inducible β-cell specific PKCΔ knockout (PKCΔ-βKO) and encapsulated in RTG-Lam and RTG-Lys for 24hrs with or without cytokines (10ng/mL TNF-α, 5ng/mL IL-1β, 100ng/mL IFN-γ). Viability, PKCΔ translocation, and PKCΔ activity were measured in encapsulated islets in situ using fluorescent biosensors or by western blot. PKCΔ-βKO islets were protected against cytokine-induced death compared to controls (p=0.003). Cytokines increased β-cell death in RTG-Lys (p<0.001), while islets encapsulated in RTG-Lam were protected against cytokine-mediated death (p<0.04). Islets encapsulated in RTG-Lam had reduced PKCΔ activity at the cell membrane compared to RTG-Lys and were also protected against cytokine-mediated increases in PKCΔ activity at the cell membrane. In summary, islet interactions with laminin protect against cytokine-induced β-cell death via downregulation of PKCΔ activity at the cell membrane. Our results suggest that loss of islet interactions with laminin, as occurs in T1D, may make islets more susceptible to cytokine-mediated death. This mechanism of β-cell death provides a novel therapeutic target to prevent β-cell death and halt the progression of T1D. Disclosure N.L. Farnsworth: None. R.A. Piscopio: None. R.K. Benninger: None. Funding JDRF (3-APF-2019-749-A-N, 1-FAC-2020-891-A-N); Colorado Clinical and Translational Sciences Institute (CO-M-19-133)

2124-P: Raptor Levels Are Critical in the Adaptation of Beta Cells to High-Fat Diet

mTORC1 signaling is a central regulator of autophagy and altered autophagic activity has been implicated in the beta-cells of patients with type 2 diabetes, and in the beta-cells of obese mice. The importance of mTORC1 signaling and Raptor in beta-cells has been recently demonstrated using raptor knock-out models (βraKO mice). These studies have shown that complete deletion of Raptor impairs beta-cell proliferation and survival of the beta-cell, and both in function and insulin processing. However, nothing is known whether reduced mTORC1 levels impair beta-cell adaptation to diabetogenic conditions. Therefore, we decided to study mice with haploinsufficiency of Raptor in beta-cells (βraHET). Mice with heterozygous deletion of Raptor in beta-cells were generated by crossing raptorf/f with Rip-Cre mice (βraHET). βraHET and control mice were fed regular chow (RC) or high-fat diet (HFD). Mice fed with RC showed no difference at the metabolic level, islets morphology, or beta-cell function. βraHET mice fed HFD for eight weeks showed an impaired Intraperitoneal Glucose Tolerance Test, fed insulin secretion and a decreased beta-cell mass, compared to control mice. Isolated islets from βraHET mice fed HFD for eight weeks showed impaired insulin secretion either measured by perifusion or static incubation compared to control islets. Next, we tested whether isolated islets from βraHET mice are more susceptible to beta-cell stressors. Beta-cell death determined by TUNEL and cleavage Caspase 3 was increased (50 and 60%) in the βraHET islets after 24 h exposure to palmitate. In addition, autophagy markers such as LC3 and p62 were increased in islets from βraHET treated with palmitate. The increase in cell death and autophagy is specific to lipotoxicity since there βraHET islets exhibited no increase in cleavage Caspase 3 in TUNEL when they were treated with proinflammatory cytokines or ER Stress inducers. In conclusion, we demonstrate that adequate levels of Raptor are critical in the adaptation of the beta-cell to HFD. Disclosure M. Blandino-Rosano: None. E. Bernal-Mizrachi: None.

Pathogenesis and remission of type 2 diabetes: what has the twin cycle hypothesis taught us?

Type 2 diabetes has been regarded a complex multifactorial disease that lead to serious health complications including high cardiovascular risks. The twin cycle hypothesis postulated that both hepatic insulin resistance and dysfunction rather than death of beta (β) cell determine diabetes onset. Several studies were carried out to test this hypothesis, and all demonstrated that chronic excess calorie intake and ectopic fat accumulation within the liver and pancreas are fundamental to the development of this disease. However, these recent research advances cannot determine the exact cause of this disease. In this review, the major factors that contribute to the pathogenesis and remission of type 2 diabetes will be outlined. Importantly, the effect of disordered lipid metabolism, characterized by altered hepatic triglyceride export will be discussed. Additionally, the observed changes in pancreas morphology in type 2 diabetes will be highlighted and discussed in relation to β cell function.

Prevention of Oxidative Stress-Induced Pancreatic Beta Cell Damage by Broussonetia Kazinoki Siebold Fruit Extract Via the ERK-Nox4 Pathway.

Pancreatic beta cells are vulnerable to oxidative stress, which causes beta cell death and dysfunction in diabetes mellitus. Broussonetia kazinoki Siebold (BK) is a widely used herbal medicine, but its potential effects against beta cell death-induced diabetes have not been studied. Therefore, we investigated the protective effect of an ethanolic extract of BK fruit (BKFE) against streptozotocin (STZ)-induced toxicity in pancreatic beta cells. Intraperitoneal injection of STZ in mice induced hyperglycemia; however, oral administration of BKFE significantly decreased the blood glucose level as well as HbA1c levels. BKFE treatment improved glucose tolerance and increased body weight in diabetic mice. Moreover, BKFE treatment resulted in increased serum insulin levels and insulin expression in the pancreas as well as decreased 4-hydroxynonenal levels induced by oxidative stress. Treatment with STZ decreased cell viability of mouse insulinoma cells (MIN6), which was blocked by BKFE pretreatment. BKFE significantly inhibited apoptotic cells and decreased the expression levels of cleaved-caspase-3 and cleaved-poly (ADP-ribose) polymerase (PARP) induced by STZ treatment. Production of reactive oxygen species in STZ-treated MIN6 cells was also significantly decreased by treatment with BKFE. Erk phosphorylation and Nox4 levels increased in STZ-treated MIN6 cells and the pancreas of mice injected with STZ and this increase was inhibited by treatment with BKFE. Inhibition of Erk phosphorylation by treatment with the PD98059 inhibitor or siRNA Erk also blocked the expression of Nox4 induced by STZ treatment. In conclusion, BKFE inhibits Erk phosphorylation, which in turn prevents STZ-induced oxidative stress and beta cell apoptosis. These results suggested that BKFE can be used to prevent or treat beta cell damage in diabetes.

Single-cell RNA sequencing of pancreatic islets reveals the complexity of autoimmune diabetes throughout its initiation and development

Abstract Type one diabetes (T1D) is a spontaneous autoimmune disease that results in pancreatic islet beta-cell death driven by diverse immune cells. However, molecular signatures of the leukocyte populations in T1D remain poorly defined. Here, using single-cell transcriptional analysis, we examined intra-islet leukocytes at various stages of disease: from its very early start, through its control phase and finally to the final effector stage. Early stages before clinical diagnosis are not accessible for human studies. We found that both CD4 and CD8 T cells showed heterogeneity at the initial stage of autoimmune inflammation and already contained effector cells. Moreover, the disease progression at all times was accompanied by regulatory sets establishing control mechanisms. In line with that, we also detected accumulation of anergic and exhaustion-associated phenotypes over time. Analysis of the islet macrophages showed substantial divergence, including novel population with anti-inflammatory and dead-cell clearance features. Autoimmune process resulted in polarization of islet macrophage into terminal pathogenic state via two-step activation program. While stage 1 marked by NF-kB activation was present in non-diabetic condition, stage 2 was inflammation-induced and had additional IFN-gamma activation. Interestingly, the final effector stages were not dominated by CD8 cytotoxic T cells but by pro-inflammatory macrophages. Overall, our analysis comprehensively describes stages of T1D on the basis of lymphoid and myeloid transcriptional activation programs, providing a detailed single-cell atlas of autoimmune diabetes initiation and development. Our findings will guide future studies in staging human T1D.

Mesenchymal stem cell-derived exosomes protect beta cells against hypoxia-induced apoptosis via miR-21 by alleviating ER stress and inhibiting p38 MAPK phosphorylation

BackgroundHypoxia is a major cause of beta cell death and dysfunction after transplantation. The aim of this study was to investigate the effect of exosomes derived from mesenchymal stem cells (MSCs) on beta cells under hypoxic conditions and the potential underlying mechanisms.MethodsExosomes were isolated from the conditioned medium of human umbilical cord MSCs and identified by WB, NTA, and transmission electron microscopy. Beta cells (βTC-6) were cultured in serum-free medium in the presence or absence of exosomes under 2% oxygen conditions. Cell viability and apoptosis were analysed with a CCK-8 assay and a flow cytometry-based annexin V-FITC/PI apoptosis detection kit, respectively. Endoplasmic reticulum stress (ER stress) proteins and apoptosis-related proteins were detected by the WB method. MiRNAs contained in MSC exosomes were determined by Illumina HiSeq, and treatment with specific miRNA mimics or inhibitors of the most abundant miRNAs was used to reveal the underlying mechanism of exosomes.ResultsExosomes derived from MSC-conditioned culture medium were 40–100 nm in diameter and expressed the exosome markers CD9, CD63, CD81, HSP70, and Flotillin 1, as well as the MSC markers CD73, CD90, and CD105. Hypoxia significantly induced beta cell apoptosis, while MSC exosomes remarkably improved beta cell survival. The WB results showed that ER stress-related proteins, including GRP78, GRP94, p-eIF2α and CHOP, and the apoptosis-related proteins cleaved caspase 3 and PARP, were upregulated under hypoxic conditions but were inhibited by MSC exosomes. Moreover, the p38 MAPK signalling pathway was activated by hypoxia and was inhibited by MSC exosomes. The Illumina HiSeq results show that MSC exosomes were rich in miR-21, let-7 g, miR-1246, miR-381, and miR-100. After transfection with miRNA mimics, the viability of beta cells under hypoxia was increased significantly by miR-21 mimic, and the p38 MAPK and ER stress-related proteins in beta cells were downregulated. These changes were reversed after exosomes were pretreated with miR-21 inhibitor.ConclusionsExosomes derived from MSCs could protect beta cells against apoptosis induced by hypoxia, largely by carrying miR-21, alleviating ER stress and inhibiting p38 MAPK signalling. This result indicated that MSC exosomes might improve encapsulated islet survival and benefit diabetes patients.