Mouse Pancreas Research Articles

1463-P: Steroidogenic Factor 1 in ß cells Moonlights as a Brake on Glucose Intolerance in Obesity

Pancreatic β cells compensate with intense insulin secretion in obese individuals, failure of which marks a vital step in progression to type 2 diabetes (T2DM) . Steroidogenic factor-1 (SF-1) is a nuclear receptor which also locates in non-steroidogenic tissues and participates in metabolic regulation. Through immunofluorescence conducted on the pancreas of obese mice, we detected SF1 in the β cells which was surprisingly absent in that of diabetic mice. Genetic deletion of SF1 in β cells predisposed mice to glucose intolerance upon high fat diet (HFD) by perturbing glucose-stimulated insulin secretion (GSIS) , while its ectopic expression by adeno-associated virus (AAV) improved GSIS. Furthermore, we illustrated that mitochondrial improvement may be the underlying mechanism via experiments such as oxygen consumption rate (OCR) , calcium detection and bioinformatics. In conclusion, SF1 played an important role in β-cell adaptation to lipotoxicity in the obese individuals, which supported SF1 as a potential target for T2DM treatment. Disclosure Y.Guo: None. L.Liu: None. Y.Li: None. Funding This work was supported by National Key Research and Development Program of China (no. 2018YFC1314100) awarded to L.-Y.B.; National Natural Science Foundation of China (no. 81870557) awarded to L.-Y.B.; Key Field Research and Development Program of Guangdong Province, China (no. 2019B020230001) awarded to L.-Y.B.; Natural Science Foundation of Guangdong Province (no. 2016A030313239 and 2014A030313018) awarded to W.-X.S.; Guangdong Basic and Applied Basic Research Foundation (no. 2020A1515010049) awarded to L.H.; and Graduate Innovative Development Program (no. 19ykyjs08) awarded to G.Y

Serological and Histological Evaluation of the Effect of Honeybee Venom on Pancreas and Liver in Diabetic Mice.

Natural toxins have been traditionally used to trigger several diseases among which bee venom (HBV) is of great importance. The present study aimed to investigate the therapeutic effects of honeybee venom (HBV) on alloxan and glucose fluid-induced Type 2 diabetes mellitus (T2DM). Therefore, a total of 20 adult laboratory male mice (Mus musculus) were selected, acclimated, and divided into four equal groups (n=5). Initially, 15 mice were fasted for 12 hrs and injected with alloxan at a single dose of 150 mg/kg of body weight. The animals were exposed to drinking glucose fluid in the morning for 4 days. Then, the blood glucose was measured. The studied animals having blood glucose of ≤200 mg/dl were considered non-diabetic and re-subjected to injecting alloxan (150 mg/kg body weight) and drinking glucose fluid for another 4 days. Four groups of mice population included, Group 1: non-diabetic and untreated with HBV, Group 2: diabetic and received no HBV as the potential therapeutic agent, Group 3: diabetic and received a low level of HBV at a dose of 0.5 mg/kg, Group 4: diabetic and received a high level of HBV at a dose of 1 mg/kg. At the end of the 35-day testing period, blood samples were tested to determine the levels of insulin, glucose, and lipid profiles [cholesterol, triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL)] using Sandwich ELISA kits. The results indicated a significant increase in blood glucose in the diabetic group compared to that of the control one, while both concentrations of HBV significantly reduced the level of blood glucose compared to that of the diabetic group. Furthermore, the level of insulin was significantly decreased in the diabetic group compared to that of the controls, while HBV significantly increased the level of insulin compared to that of the diabetic group. Moreover, the diabetic mice demonstrated a significant increase in the concentration of cholesterol and TG compared to that of control mice which were significantly reversed in response to HBV treatment. The level of HDL was significantly decreased in the diabetic group compared to that of the control group which was modulated by treatment, while no significant differences were seen between all the studied groups regarding the level of LDL. Histological examination of diabetic mice revealed a significant alteration in acinar cells and destruction of β-cells of pancreatic sections with marked lacerations in the liver extended to all structures of the organ. The present study concluded that HBV could be a potential therapeutic agent to prevent and manage diabetes and its complication.

The role of CXCR1/2 dual inhibitor ladarixin on tumor burden and immunotherapy response in pancreatic cancer.

e16304 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies for which few effective pharmacological treatments are currently available. Immunotherapy has revolutionized the treatment of several solid tumors, yet the results obtained in PDAC have been disappointing. Although PDAC is generally considered an immune “cold” cancer, different PDAC subtypes have diverse immune tissue microenvironment (TME) components, including tumor-associated macrophages (TAMs), that might differentially influence immunotherapy responsiveness and patient survival. TAM population is plastic, switching rapidly from a pro- to an anti-tumoral behaviour, based on specific cytokines in TME. A role for the chemokines/CXCR axis in inducing M1 polarization has been proposed in several tumors, and, consequently, the potential advantage of combining CXCR inhibitors with immune check-point inhibitors. Here, we evaluated the effectiveness of the CXCR1/2 inhibitor ladarixin, alone or in combination with anti-PD-1, on immune tolerance against PDAC and on tumor growth. Methods: A set of preclinical models were obtained by engrafting mouse PDAC-derived cell lines into the pancreas of recipient syngeneic immune-competent mice, as well as by orthotopically transplanting patient derived PDAC tumor into human immune system reconstituted (HIR) mice (Hu-NSG-CD34+). Tumor bearing mice were randomly assigned to receive vehicles, ladarixin, and anti-PD-1 alone or in combination with ladarixin, to evaluate the effect of treatment on tumor growth and mice survival. This work is supported by AIRC and Italian Ministry of Health grants. Results: CXCR1/2 inhibition by ladarixin reverted in vitro tumor-mediated M2 polarization and migration of Bone Marrow-Derived Macrophages. Ladarixin as a single agent significantly reduced tumor burden in cancer derived graft (CDG) models with high-immunogenic potential and significantly increased the efficacy of ICI in non-immunogenic CDG models that did not respond to anti-PD-1 treatment. In a HIR mouse model bearing the immunogenic subtype of human PDAC, ladarixin showed high efficacy as a single agent and the ability to increase the antitumor effects of anti-PD-1 with a statistically significant reduction of tumor volume compared to control and to single treatments (ctr vs lad, p = 0.0077; ctr vs comb, p = 0.0098; lad vs comb: 0.002; anti-PD-1 vs comb, p = 0.036). Moreover, ladarixin both as monotherapy and in combination setting increased survival (median survival: ctr 49.5 vs lad 74.5 days, p = 0.044; ctr 49.5 vs comb 150 days, p = 0.0108; anti-PD-1 57 vs comb 150 days, p = 0.046; lad 74.5 vs comb 150 days, p = 0.047). Conclusions: Ladarixin in combination with anti-PD-1 might represent an effective approach for the treatment of PDAC, converting a protumoral into an immunopermissive microenvironment in PDAC subtypes usually refractory to immunotherapy.

252-LB: GRK2 Participates in Islet Function and Glucose-Stimulated Insulin Secretory Responses

Insulin deficiency is central to diabetes and diabetes-related cardiac dysfunction. GPCRs are known modulators of insulin secretion and a main pharmacological target in various tissues, including the heart. GPCR kinase 2 (GRK2) phosphorylates activated GPCRs, targeting receptors for recycling or degradation. Notably, we and others have shown that GRK2 can also localize to the cardiac mitochondria where it participates in substrate utilization, particularly in response to cellular stress. GRK2 is downregulated in the pancreas of diabetogenic mice, and we have shown that pancreatic loss of GRK2 impairs insulin secretion in normal and high fat diet. Mice with pancreatic-specific GRK2 KO showed glucose intolerance (AUC WT 8691 vs. KO 14766 mg/dl*min, n=22/group, p<.05) due to significant decrease in insulin secretion following glucose administration (after 15 minutes WT 2.35 vs. KO 1.616 ng/ml, N=16-19/group, P<.0001) without changes in islet size or cell distribution. Using a model of high fat diet, we observed an impairment in glucose-stimulated insulin release in pancreatic GRK2 KO animals when compared to control animals without significant changes in fasting insulin secretion or insulin sensitivity. To further dissect the role of islet GRK2 in α- or β-cells, we have constructed two novel mouse models of inducible cell type-specific GRK2 KO. Our preliminary studies reveal that β-cell GRK2 deficient animals are prone to glucose intolerance and decreased glucose-induced insulin secretion when exposed to high fat high sucrose diet. This suggests that GRK2 plays a role in islet adaptation to metabolic challenge. Further studies will delineate the mechanistic role of GRK2 in mediating metabolism and calcium influx in β-cells regulating insulin secretion, and subsequently the impact to the heart in the presence or absence of TAC. Our studies will provide new GRK2 mechanistic insight that could be explored as new pharmacological strategies to delay diabetes disease progression. Disclosure J. W. Snyder: None. S. K. Montgomery: None. N. M. Doliba: None. J. Roman: None. Y. Tian: None. P. Y. Sato: None. W. L. Holland: None. R. Choi: None. Funding National Institutes of Health (1R56HL149887) ; University of Pennsylvania Diabetes Research Center Pilot and Feasibility Grant (P30-DK019525) ; American Heart Association Scientist Development Grant (17SDG33660407)

368-OR: Activation of a Yap1-Dependent Mesenchymal Program Promotes ß-Cell Failure

A critical contributor to the pathogenesis of type 2 diabetes is the failure of insulin-secreting β-cells due to loss of cell number or identity. However, the signaling pathways that trigger these events remain unclear. While β-cells are of endodermal origin, recent studies show endocrine cells that coexpress the mesenchymal marker vimentin in the pancreas with diabetes, although the functional features of these cells are not known. Here, we identify an epithelial-to-mesenchymal (EMT) -like program that is activated in islets of mice and humans with type 2 diabetes. Characteristics of this EMT-like program include increased protein and mRNA abundance of the EMT transcription factor Snail and the mesenchymal adhesion molecule N-cadherin. Lineage-tracing techniques demonstrate that this mesenchymal program is initiated in β-cells that become multi-hormonal, a hallmark of dysfunctional endocrine cells. Interestingly, EMT-like cells minimally express Aldh1a3, a marker of dedifferentiated β-cells. To determine the mechanism driving EMT-like activation in β-cells, we tested stressors that phenocopy the effects of diabetes in islets. Treatment of islets with mitochondrial decouplers as well as oxidative stress increased expression of EMT markers. The transcription factor Yap1 is a known inducer of EMT. While Yap1 is expressed at minimal levels in β-cells, its abundance increased in db/db islets and in primary cells with impaired mitochondrial function. Importantly, chemical inhibition of Yap1 in db/db islets reversed activation of EMT-like factors. Yap1 inhibition also improved insulin secretion. Taken together, we found activation of an EMT-like program in the endocrine pancreas of mice and humans with type 2 diabetes. This program is dependent on increases in Yap1 due to oxidative stress and impaired mitochondrial function, and results in disrupted β-cell function. Disclosure W.Mckimpson: n/a. N.Hoque: None. J.Son: None. A.Y.Lee: None. D.Accili: Advisory Panel; Lilly Diabetes. Funding NIDDK (1K01DK121873)

Alleviation of oxidative stress in pancreatic tissue of hyperglycemic mice by Lactiplantibacillus plantarum SCS4.

This study aimed to investigate the effect of Lactiplantibacillus plantarum SCS4 in alleviating oxidative stress in the pancreatic tissue of hyperglycemic mice. A total of 90 six-week-old specific pathogen-free male Kunming mice were randomly divided into six groups [normal group (NG), blank control group (MG), phosphate-buffered saline (PBS) control group (CG), SCS4 first control group (DT1), SCS4 second control group (DT2), and SCS4 third control group (DT3)]. Except the NG group, in the other five groups, streptozotocin (STZ) was intraperitoneally injected for five consecutive days to establish a hyperglycemia model; the concentration of STZ was 50 mg/kg (bw). After successful modeling, DT1, DT2, and DT3 mice were administered 0.2ml of L. plantarum SCS4 bacterial solution (1010 colony forming unit/ml), the cellular content of L. plantarum SCS4, and the inactivated cellular content of L. plantarum SCS4, respectively. Furthermore, 0.2ml of PBS was given to mice in the CG group for control. The levels of insulin (INS), reactive oxygen species (ROS), malondialdehyde (MDA), and antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were measured by enzyme-linked immunosorbent assay, and the morphology of the pancreas was observed. The results showed that after 10 weeks of gavage treatment, the level of INS in the DT3 group significantly increased to 6.36 mIU/L compared with that in the MG group (p < .05). Meanwhile, the levels of ROS and MDA of DT3 returned to normal levels of 291.07 IU/ml and 4.29 mnol/L, respectively. The activities of various antioxidant enzymes increased. The levels of SOD, CAT, and GPx in DT3 were the closest to the levels in NG. In addition, the pathological sections showed that the degree of pancreatic tissue lesions was relatively more severe in the MG group than in the NG group. The degree of pancreatic tissue lesions was relatively less severe in the DT2 group than in the MG group, and no significant lesion was seen in the DT3 group. Our results indicated that the inactivated bacterial content of L. planetarium SCS4 was more effective in improving oxidative stress in the pancreas of hyperglycemic mice. PRACTICAL APPLICATIONS: L.plantarum SCS4 was separated from fermented sausage in Sichuan. This study indicated that inactivated bacterial content of L. planetarium SCS4 was more effective in improving oxidative stress in the pancreas of hyperglycemic mice. The results suggested that lactic acid bacteria from traditional foods with ability of improving oxidative damage, which can be applied in food nutrition and related fields to make people with good dietary habits and prevent the occurrence of chronic diseases such as type II diabetes effectively.

GFP Tagged VSELs Help Delineate Novel Stem Cells Biology in Multiple Adult Tissues.

Various types of stem cells are being researched upon to exploit their potential for regenerative medicine including pluripotent human embryonic stem (hES) cells derived from spare human embryos, induced pluripotent stem (iPS) cells by reprogramming somatic cells to a pluripotent state and multipotent mesenchymal stem/stromal cells (MSCs) obtained in vitro from multiple tissues. More than 50 independent groups have reported another novel population of pluripotent stem cells in adult tissues termed very small embryonic-like stem cells (VSELs). VSELs are developmentally linked to primordial germ cells, which rather than giving rise to the germ cells and later ceasing to exist, survive throughout life in multiple organs along with tissue-specific adult stem cells better described as lineage-restricted, tissue-committed progenitors with limited plasticity. VSELs survive total body irradiation in bone marrow, oncotherapy in the gonads, bilateral ovariectomy in the uterus and partial pancreatectomy in the pancreas of mice and participate in the regeneration of multiple organs under normal physiological conditions. VSELs and tissue-specific progenitor cells work together in a subtle manner, maintain life-long tissue homeostasis and their dysfunction leads to various pathologies including cancer. However, due to their quiescent state, VSELs have invariably eluded lineage-tracing studies reported so far. Present article reviews novel insights into VSELs biology and how VSELs enriched from GFP (green fluorescent protein) mice have enabled to delineate their role in various biological processes in vivo. VSELs biology needs to be understood in-depth as this alone will help evolve the field of regenerative medicine and win the war against cancer.

Combination of anti-CD25 antibody and poly I:C treatment in pregnant NOD mice may be used as 'pregnancy-related' type 1 diabetes model.

ABSTRACTAims/IntroductionSome women develop type 1 diabetes during pregnancy or immediately after delivery. However, the underlying pathophysiology remains largely unknown, probably because of the lack of a suitable animal model. In this study, we administered pregnant NOD mice with an anti‐CD25 antibody to reduce regulatory T cells along with poly I:C and examined the onset of diabetes.Materials and MethodsAnti‐CD25 antibody and poly I:C were intraperitoneally administered to mated female NOD mice. Mice delivered within 3 weeks after the treatment, and the onset of diabetes during pregnancy or within 6 weeks after delivery was examined. Some mice were killed 1 week after treatment, and their spleen and pancreas were excised to examine the expression levels of cytokines and for histological examination.ResultsHalf of the mice treated with anti‐CD25 antibody plus poly I:C developed diabetes, as compared with none of the poly I:C‐injected mice (P < 0.05). The ratios of interleukin‐18/forkhead box P3 and granzyme B/forkhead box P3 were higher in the pancreas of anti‐CD25 antibody plus poly I:C‐treated mice than in the pancreas of control mice. The insulitis score decreased in the pancreas of anti‐CD25 antibody plus poly I:C‐injected pregnant NOD mice.ConclusionsWe describe the use of anti‐CD25 antibody to reduce regulatory T cells and poly I:C as a Toll‐like receptor 3 stimulator to mimic viral infection in a pregnant NOD mouse, which can be used as a model of ‘pregnancy‐related’ type 1 diabetes.

Hypoglycemic Effect of Nobiletin via Regulation of Islet β-Cell Mitophagy and Gut Microbiota Homeostasis in Streptozocin-Challenged Mice.

Nobiletin is a natural nutrient (or polymethoxyflavonoid) in orange peels exerting a preventive effect against metabolic diseases. However, there are very few reports on the hypoglycemic effect of nobiletin. In the present study, the hypoglycemic effect of nobiletin was investigated using NIT-1 cells and streptozocin (STZ)-challenged mouse models. Our results indicated that nobiletin could significantly suppress the high blood glucose in STZ-challenged mice. In addition, nobiletin could effectively activate the mitophagy and inhibit the inflammatory pathways in NIT-1 cells. The mitochondria membrane potential dysbiosis induced by glucotoxicity in NIT-1 cells was restored after treatment by nobiletin. Further investigation revealed that the hypoglycemic effect of nobiletin was mainly through regulation of gut microbiota dysbiosis, activation of mitophagy flux, inhibition of inflammasome expression, and restoration of islet morphological destruction in the pancreas of STZ-challenged mice. Our study revealed that nobiletin could be used as a functional food or drug candidate for the treatment of diabetes.

Abrogating regulatory T cells overcomes tumor-specific T cell exhaustion and prevents metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related deaths and has a dismal 5-year survival rate of 10 percent. PDA lethality is attributed to late diagnosis, early metastasis, and therapeutic resistance. Metastasis can occur before the development of histologically detectable tumors and is a leading cause of cancer-related deaths. We identified that pancreatic tumor cells derived from mice that resist immunotherapy (e.g., tumor escape variants, TEV) and re-implanted into the pancreas of syngeneic and immunocompetent mice rapidly metastasize, reflecting the pathogenesis of human PDA. We show that TEVs retain the targeted tumor antigen, and despite a defect in IFNgamma-inducible MHC class I upregulation, TEVs remain sensitive to tumor antigen specific T cell-mediated lysis in vitro, suggesting TEVs may confer unique qualities in vivo to resist T cell killing. Using a peptide:MHC tetramer to identify the tumor-specific CD8 T cells, we identified that intratumoral T cells in EV tumors have increased Granzyme B production and a reduction in prototypical exhaustion markers PD1, Lag3, and Tox, perhaps due to reduced MHC class I signaling. Notably, primary tumors from TEVs were significantly enriched for Foxp3+ Tregs as compared to parental tumors. Using a genetic model, we identified that Treg depletion resulted in a drastic reduction in tumor burden and metastasis and improved tumor-specific T cell function in TEV tumors. Tumor-cell intrinsic changes driving Treg accumulation in TEVs will be discussed. In summary, Tregs are key drivers of both T cell exhaustion and immunosuppression in pancreatic cancer and may prove a valuable clinical target for tumors that evade immune checkpoint blockade. Supported by grants from NIH (R01 CA249393)

Type I Diabetes induced by OT-I T-Cells requires ITK-mediated TCR signaling.

Abstract Activation of CD8+ T-cells induces changes in T cell trafficking, promoting effector T cell migration into tissues. ITK, a tyrosine kinase activated by TCR signaling, has previously been shown to be required for CD8+ T cell migration into the gastrointestinal tract during viral infection. This finding prompted us to investigate whether ITK signaling was important for effector T cell trafficking into non-infected tissue, as might occur in autoimmune disease. Using the RIP-mOVA mouse model, in which the OVA protein is expressed in the β-islet cells of the pancreas, we examined the ability of WT versus ITK-deficient OT-I T cells to infiltrate the pancreas inducing Type I diabetes. To test this, WT or ITK-deficient OT-I cells were adoptively transferred into RIP-mOVA recipients. Mice were immunized with OVA peptide plus LPS, and monitored for urine glucose. We also examined OT-I cell numbers in the pancreatic draining lymph node and in the pancreas 72hr post-immunization. We found that in the absence of ITK, OT-I cells were impaired in their ability to induce diabetes, with only ~1/3 of the recipients progressing to disease; in contrast 100% of recipients receiving WT OT-I cells developed Type I diabetes. In addition, the numbers of infiltrating ITK-deficient OT-I cells in the pancreas was significantly reduced compared to the numbers of WT OT-I cells detected. These findings indicate that ITK has a key role in generating pathogenic tissue-infiltrating CD8+ T cells capable of migration into β-islets of the mouse pancreas. While exploration of the mechanism underlying this impaired migration needs further investigation, these data provide support for therapeutic potential of an ITK inhibitor in patients with tissue-specific autoimmune diseases. Supported by NAID TRC SIGNLG

Impaired regulation of PMCA activity by defective CFTR expression promotes epithelial cell damage in alcoholic pancreatitis and hepatitis.

Alcoholic pancreatitis and hepatitis are frequent, potentially lethal diseases with limited treatment options. Our previous study reported that the expression of CFTR Cl- channel is impaired by ethanol in pancreatic ductal cells leading to more severe alcohol-induced pancreatitis. In addition to determining epithelial ion secretion, CFTR has multiple interactions with other proteins, which may influence intracellular Ca2+ signaling. Thus, we aimed to investigate the impact of ethanol-mediated CFTR damage on intracellular Ca2+ homeostasis in pancreatic ductal epithelial cells and cholangiocytes. Human and mouse pancreas and liver samples and organoids were used to study ion secretion, intracellular signaling, protein expression and interaction. The effect of PMCA4 inhibition was analyzed in a mouse model of alcohol-induced pancreatitis. The decreased CFTR expression impaired PMCA function and resulted in sustained intracellular Ca2+ elevation in ethanol-treated and mouse and human pancreatic organoids. Liver samples derived from alcoholic hepatitis patients and ethanol-treated mouse liver organoids showed decreased CFTR expression and function, and impaired PMCA4 activity. PMCA4 co-localizes and physically interacts with CFTR on the apical membrane of polarized epithelial cells, where CFTR-dependent calmodulin recruitment determines PMCA4 activity. The sustained intracellular Ca2+ elevation in the absence of CFTR inhibited mitochondrial function and was accompanied with increased apoptosis in pancreatic epithelial cells and PMCA4 inhibition increased the severity of alcohol-induced AP in mice. Our results suggest that improving Ca2+ extrusion in epithelial cells may be a potential novel therapeutic approach to protect the exocrine pancreatic function in alcoholic pancreatitis and prevent the development of cholestasis in alcoholic hepatitis.

Co-Crystal of Rosiglitazone With Berberine Ameliorates Hyperglycemia and Insulin Resistance Through the PI3K/AKT/TXNIP Pathway In Vivo and In Vitro.

Background: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by insulin resistance and hyperglycemia. This study examined the effect and elucidated the mechanism of improvement of hyperglycemia and insulin resistance by a co-crystal of rosiglitazone with berberine (RB) in high-sugar high-fat diet (HSHFD)-induced diabetic KKAy mice. Methods: Diabetic KKAy mice were randomly divided into seven groups: KKAy model control group (DM control) treated with 3% sodium carboxymethyl cellulose; RB groups, administered daily with RB 0.7 mg/kg (RB-L), 2.11 mg/kg (RB-M), or 6.33 mg/kg (RB-H); positive control groups, administered daily with rosiglitazone 1.04 mg/kg (RSG), berberine 195 mg/kg (BBR), or combination of 1.04 mg/kg RSG and 1.08 mg/kg BBR (MIX). Test compounds were administered orally for 8 weeks. Non-diabetic C57BL/6J mice were used as normal control (NC). Blood glucose, food intake, body weight, glucose-lipid metabolism, and pathological changes in the pancreas and liver were examined. We further evaluated the mechanism of action of RB in C2C12 and HepG2 cells stimulated with high glucose and palmitate. Results: RB treatment improved glucolipid metabolism and insulin resistance in diabetic KKAy mice. RB reduced blood glucose levels, white fat index, plasma triglyceride (TG), low-density lipoprotein (LDL), gastric inhibitory peptide (GIP), and insulin levels, increased the levels of plasma glucagon-like peptide-1 (GLP-1), high-density lipoprotein (HDL), and glycogen content in the liver and muscle; and improved oral glucose tolerance test (OGTT), insulin tolerance test (ITT), and pathological changes in the pancreas and liver of KKAy mice. Moreover, RB upregulated p-PI3K and p-AKT levels and reduced TXNIP expression in KKAy mice and in HepG2 and C2C12 cells. Conclusion: These data indicate that RB ameliorates insulin resistance and metabolic disorders, and the mechanism might be through regulating the PI3K/AKT/TXNIP signaling pathway . Thus, the co-crystal drug RB may be considered as a potential antidiabetic agent for future clinical therapy.

Misfolding-induced chronic pancreatitis in CPA1 N256K mutant mice is unaffected by global deletion of Ddit3/Chop

Genetic mutations in pancreatic digestive enzymes may cause protein misfolding, endoplasmic reticulum (ER) stress and chronic pancreatitis. The CPA1 N256K mouse model carries the human p.N256K carboxypeptidase A1 (CPA1) mutation, a classic example of a pancreatitis-associated misfolding variant. CPA1 N256K mice develop spontaneous, progressive chronic pancreatitis with moderate acinar atrophy, acinar-to-ductal metaplasia, fibrosis, and macrophage infiltration. Upregulation of the ER-stress associated pro-apoptotic transcription factor Ddit3/Chop mRNA was observed in the pancreas of CPA1 N256K mice suggesting that acinar cell death might be mediated through this mechanism. Here, we crossed the CPA1 N256K strain with mice containing a global deletion of the Ddit3/Chop gene (Ddit3-KO mice) and evaluated the effect of DDIT3/CHOP deficiency on the course of chronic pancreatitis. Surprisingly, CPA1 N256K x Ddit3-KO mice developed chronic pancreatitis with a similar time course and features as the CPA1 N256K parent strain. In contrast, Ddit3-KO mice showed no pancreas pathology. The observations indicate that DDIT3/CHOP plays no significant role in the development of misfolding-induced chronic pancreatitis in CPA1 N256K mice and this transcription factor is not a viable target for therapeutic intervention in this disease.

CARD9 Mediates Pancreatic Islet Beta-Cell Dysfunction Under the Duress of Hyperglycemic Stress.

Background/Aims:Published evidence implicates Caspase recruitment domain containing protein 9 (CARD9) in innate immunity. Given its recently suggested roles in obesity and insulin resistance, we investigated its regulatory role(s) in the onset of islet beta cell dysfunction under chronic hyperglycemic (metabolic stress) conditions.Methods:Islets from mouse pancreas were isolated by the collagenase digestion method. Expression of CARD9 was suppressed in INS-1 832/13 cells by siRNA transfection using the DharmaFect1 reagent. The degree of activation of Rac1 was assessed by a pull-down assay kit. Interactions between CARD9, RhoGDIβ and Rac1 under metabolic stress conditions were determined by co-immunoprecipitation assay. The degree of phosphorylation of stress kinases was assessed using antibodies directed against phosphorylated forms of the respective kinases.Results:CARD9 expression is significantly increased following exposure to high glucose, not to mannitol (both at 20 mM; 24 hrs.) in INS-1 832/13 cells. siRNA-mediated knockdown of CARD9 significantly attenuated high glucose-induced activation of Rac1 and phosphorylation of p38MAPK and p65 subunit of NF-κB (RelA), without significantly impacting high glucose-induced effects on JNK1/2 and ERK1/2 activities. CARD9 depletion also suppressed high glucose-induced CHOP expression (a marker for endoplasmic reticulum stress) in these cells. Co-immunoprecipitation studies revealed increased association between CARD9-RhoGDIβ and decreased association between RhoGDIβ-Rac1 in cells cultured under high glucose conditions.Conclusion:Based on these data, we conclude that CARD9 regulates activation of Rac1-p38MAPK-NFκB signaling pathway leading to functional abnormalities in beta cells under metabolic stress conditions.

Hormonal and enzymatic analysis for pancreas of diabetic and obese mice in Iraq

Hormonal and enzymatic analysis for pancreas of diabetic and obese mice in Iraq

205 Targeting Homologous Repair to Overcome Genotoxic Therapy Resistance in Pancreatic Cancer

OBJECTIVES/GOALS: Pancreatic ductal adenocarcinoma (PDAC) is a relatively radioresistant disease, and inhibition of DNA homologous recombination (HR) repair in combination with radiation therapy (RT) is a potentially attractive strategy to overcome radioresistance. We have found that the expression of the HR protein RAD18 is upregulated in PDAC cells. METHODS/STUDY POPULATION: Standard clonogenic assays, γH2aX foci staining, HR-GFP reporter assay, and western blot analysis of DNA damage response proteins were performed in MIA-PaCa2 (MP2) and PANC-1 cells following knockdown of RAD18 in cells via short hairpin RNA (shRNA). Drug targeting of RAD18 was achieved through the use of a USP-7 inhibitor, P5091. Cells with or without stable knockdown of RAD18 were implanted orthotopically in the pancreas of athymic nude mice and treated with sham radiation or radiation to a dose of 20 Gy in 5 daily fractions once tumors reached 100-150 mm3. RESULTS/ANTICIPATED RESULTS: Stable knockdown of RAD18 in MP2 and PANC-1 resulted in decreased radiation clonogenic survival in vitro (dose enhancement factor (DEF)=1.52 and 1.51, respectively), decreased DNA repair after radiation as measured by the increased number of γH2aX nuclear foci assay at 6, 12, and 24 hours (all p&lt;0.05), decreased HR activation following DNA damage via an HR-GFP reporter assay (p=0.039), and increased tumor growth delay following radiation in vivo (p&lt;0.001). P5091 treatment of both MP2 and PANC-1 resulted in efficient knockdown of RAD18, which was confirmed through western blotting, qRT-PCR, and luciferase reporter assays. P5091 increased radiosensitization, yH2aX nuclear foci remained elevated at 12 and 24 hours (p&lt;0.05), and HR repair was also reduced (p=0.014). DISCUSSION/SIGNIFICANCE: Herein, we show the HR repair protein RAD18, and that modulation of RAD18 expression correlates with in vitro and in vivo radiosensitization through altered HR-mediated DNA repair. USP7 inhibition successfully reduced RAD18 expression and resulted in enhanced radiosensitization.

Metabolic Regulation of Hepatitis B Virus Infection in HBV-Transgenic Mice.

Hepatitis B virus (HBV) infection is a worldwide health burden. Metabolomics analysis has revealed HBV-induced metabolism dysregulation in liver tissues and hepatocytes. However, as an infectious disease, the tissue-specific landscape of metabolic profiles of HBV infection remains unclear. To fill this gap, we applied untargeted nuclear magnetic resonance (NMR) metabolomic analysis of the heart, liver, spleen, lung, kidney, pancreas, and intestine (duodenum, jejunum, ileum) in HBV-transgenic mice and their wild-type littermates. Strikingly, we found systemic metabolic alterations induced by HBV in liver and extrahepatic organs. Significant changes in metabolites have been observed in most tissues of HBV-transgenic mice, except for ileum. The metabolic changes may provide novel therapeutic targets for the treatment of HBV infection. Moreover, tissue-specific metabolic profiles could speed up the study of HBV induced systemic metabolic reprogramming, which could help follow the progression of HBV infection and explain the underlying pathogenesis.

Adrenomedullin and its receptors are expressed in mouse pancreatic β-cells and suppresses insulin synthesis and secretion.

Gestational diabetes mellitus (GDM) is associated with defective pancreatic β-cell adaptation in pregnancy, but the underlying mechanism remains obscure. Our previous studies demonstrated that GDM women display increased plasma adrenomedullin (ADM) levels, and non-obese GDM mice show decreased serum concentrations of insulin and the number of β-cells in pancreas islets. The aims of this study is to examine if ADM and its receptors are expressed in female mouse pancreas, and if so, whether insulin secretion is regulated by ADM in mouse β-cell line, NIT-1 cells and isolated mouse pancreatic islets. Present study shows that ADM and its receptor components CRLR, RAMPs are present in mouse pancreatic islets and co-localized with insulin. The expressions of ADM, CRLR and RAMP2 in islets from pregnant mice are reduced compared to that of non-pregnant mice. NIT-1-β cells express ADM and its receptor mRNA, and glucose dose-dependently stimulates expressions. Furthermore, ADM inhibits NIT-1-β cell growth, and this inhibition is reversed by ADM antagonist, ADM22-52. The glucose-induced insulin secretion was suppressed by ADM in NIT-1-β cells and isolated pancreatic islets from pregnant mice. These inhibitory effects are accompanied by upregulation of endoplasmic reticulum (ER) stress biomarker genes in NIT-1-β cells. This study unveils that reduced ADM and its receptors may play a role in β-cell adaptation during pregnancy, while increased plasma ADM in GDM may contribute to the β-cells dysfunction, and blockade of ADM may reverse β-cell insulin production.

CRISPR somatic genome engineering and cancer modeling in the mouse pancreas and liver.

Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.