Expression In Pancreatic Tissues Research Articles

Terazosin, a repurposed GPR119 agonist, ameliorates mitophagy and β-cell function in NAFPD by inhibiting MST1-Foxo3a signalling pathway.

GPR119 agonists are being developed to safeguard the function of pancreatic β-cells, especially in the context of non-alcoholic fatty pancreas disease (NAFPD) that is closely associated with β-cell dysfunction. This study aims to employ a drug repurposing strategy to screen GPR119 agonists and explore their potential molecular mechanisms for enhancing β-cell function in the context of NAFPD. MIN6 cells were stimulated with palmitic acid (PA), and a NAFPD model was established in GPR119-/- mice fed with a high-fat diet (HFD). Terazosin, identified through screening, was utilized to assess its impact on enhancing β-cell function via the MST1-Foxo3a pathway and mitophagy. Terazosin selectively activated GPR119, leading to increased cAMP and ATP synthesis, consequently enhancing insulin secretion. Terazosin administration improved high blood glucose, obesity, and impaired pancreatic β-cell function in NAFPD mice. It inhibited the upregulation of MST1-Foxo3a expression in pancreatic tissue and enhanced damaged mitophagy clearance, restoring autophagic flux, and improving mitochondrial quantity and structure in β-cells. Nevertheless, GPR119 deficiency negated the positive impact of terazosin on pancreatic β-cell function in NAFPD mice and abolished its inhibitory effect on the MST1-Foxo3a pathway. Terazosin activates GPR119 on the surface of pancreatic β-cells, enhancing mitophagy and alleviating β-cell dysfunction in the context of NAFPD by suppressing the MST1-Foxo3a signalling pathway. Terazosin could be considered a priority treatment for patients with concomitant NAFPD and hypertension.

Effects of 3.5-GHz radiofrequency radiation on energy-regulatory hormone levels in the blood and adipose tissue.

In recent years exposure of living beings to radiofrequency radiation (RFR) emitted from wireless equipment has increased. In this study, we investigated the effects of 3.5-GHz RFR on hormones that regulate energy metabolism in the body. Twenty-eight rats were divided into four groups: healthy sham (n = 7), healthy RFR (n = 7), diabetic sham (n = 7), and diabetic RFR (n = 7). Over a month, each group spent 2 h/day in a Plexiglas carousel. The rats in the experimental group were exposed to RFR, but the sham groups were not. At the end of the experiment, blood and adipose tissues were collected from euthanized rats. Total antioxidant, total oxidant, hydrogen peroxide, ghrelin, nesfatin-1, and irisin were determined. Insulin expression in pancreatic tissues was examined by immunohistochemical analysis. Whole body specific absorption rate was 37 mW/kg. For the parameters analyzed in blood and fat, the estimated effect size varied within the ranges of 0.215-0.929 and 0.503-0.839, respectively. The blood and adipose nesfatin-1 (p = 0.002), blood and pancreatic insulin are decreased, (p = 0.001), gherelin (p = 0.020), irisin (p = 0.020), and blood glucose (p = 0.040) are increased in healthy and diabetic rats exposed to RFR. While nesfatin-1 are negatively correlated with oxidative stress, hyperglycemia and insulin, ghrelin and irisin are positively correlated with oxidative stress and hyperglycemia. Thus, RFR may have deleterious effects on energy metabolism, particularly in the presence of diabetes.

Impact of High-Intensity Interval Training on GLP-1R/ PKBα Axis in Pancreatic Tissue of Diabetes Rats Induced by High-Fat Diet and STZ

Objective: Apart from hormonal factors and oxidative stress, insulin synthesis is strongly dependent on transcription factors in the pancreas. The aim of the present study was to assess the impact of high-intensity interval training (HIIT) on genes affecting insulin synthesis in diabetic obese rats.
 Materials and Methods: Type 2 diabetes (T2D) was induced by a 6-week high-fat diet (HFD) and intraperitoneal injection of streptozotocin (25 mg /kg) in 14 male Wistar rats (10 week old, 220±10 g). Rats with fasting glucose levels between 400 and 150 were considered T2D. The diabetic rats were randomly assigned to exercise (HIIT: 6 weeks/5 sessions weekly, n= 7) or control (n= 7) groups. Forty-eight hours after the intervention, fasting GLP-1R and PKBα gene expression in pancreatic tissue and plasma insulin and glucose levels were compared between the groups. Data were compared by independent t-test used to compare variables, version 22 between groups. A P< 0.05 was considered significant.
 Results: HIIT led to significant increase in PKBα gene expression (P: 0.001) and insulin (P: 0.031) and decreases in glucose concentration (P: 0.001) compared with the control group. No change was observed in the GLP-1R gene expression response to HIIT (P: 0.093).
 Conclusion: HIIT is associated with increased serum insulin levels in T2D obese rats. Despite no change in GLP-1R, this improvement is probably rooted in increased expression PKBα in pancreas in response to this type of exercise training

ABCA12 Promotes Proliferation and Migration and Inhibits Apoptosis of Pancreatic Cancer Cells Through the AKT Signaling Pathway

Background: As a malignant tumor, pancreatic cancer is difficult to detect in its early stage. Pancreatic cancer progresses rapidly and has a short survival time. Most cases have metastasized to distant organs before diagnosis. The mechanism of induction of pancreatic cancer is not fully understood. Methods: In this study, bioinformatics predicted ATP binding cassette subfamily A member 12 (ABCA12) expression in pancreatic tissues and performed survival analysis, risk assessment, and enrichment analysis. The expression of ABCA12 in 30 pairs of clinical samples was detected by immunohistochemistry and we analyzed its correlation with clinical information. Both reverse transcription polymerase chain reaction (RT–PCR) and western blot analysis were used to detect mRNA and protein expression in cell lines. Two different siRNAs and SW1990 cell line were used to construct pancreatic cancer cell models with ABCA12 knockdown. Cell viability was evaluated by cell counting kit-8 (CCK-8) and EdU proliferation assays. Wound healing assays and Transwell assays were used to measure the ability of cell migration and invasion. Flow cytometry was used to investigate the effect of ABCA12 on the proliferation cycle and apoptosis of pancreatic cancer. Western blot analysis detected changes in apoptosis, migration, and other pathway proteins in SW1990 cells after transfection. Results: ABCA12 is highly expressed in pancreatic cancer tissues and cells. After ABCA12 was knocked down, the proliferation, invasion, and migration of SW1990 cells were significantly reduced, and apoptosis was increased. The changes in pathway proteins suggested that ABCA12 may regulate the progression of pancreatic cancer through the AKT pathway. Conclusion: We found that ABCA12 is differentially expressed in pancreatic tissues and cells. ABCA12 can also affect the biological behavior of pancreatic cancer cells effectively, which may serve as a new target for pancreatic cancer diagnosis and treatment.

Antidiabetic effects of Scrophularia striata ethanolic extract via suppression of Pdx1 and Ins1 expression in pancreatic tissues of diabetic rats

One of the factors that causes severe metabolic imbalance and abnormal changes in many tissues, especially in the pancreas, is the pathological disease of diabetes mellitus. Therefore, in this study, the therapeutic effects of Scrophularia striata were investigated using an animal model in the control of diabetic injury and pancreatic complications caused by diabetes. A total of 66 rats (weight 220–250 g) were randomly divided into: Healthy Control group (rats without diabetes receiving Propylene glycol as solvent); Diabetic control group; 3 experimental healthy groups (receiving the extract with doses of 100, 200 and 400 mg/kg bw/day); 3 treatment groups; and3 pretreatment groups. Diabetes was induced in rats by intraperitoneal STZ (60 mg/kg bw). FBS, HbA1c and insulin were measured after 4 weeks. Pdx1 and Ins1 gene expression was assessed by RT-PCR. The histological evaluation was also performed with H&E staining. The data were analyzed by SPSS ver20 using ANOVA and Tukey tests. By treatment with S. striata ethanolic extract, these factors were close to the normal range. The expression of the Pdx1 and Ins1 genes increased in the treated rats with S. striata extract. Analysis of the obtained data indicates the effect of S. striata in improving the complications of diabetes in rats and can be considered for therapeutic purposes.

NLRP-3 Expression in Pancreatic Tissue Following Moderate and High Intensity Interval Training with Liposomal Alpha-Lipoic Acid Supplement in Diabetic Rats

Introduction: Activation of NLRP-3 Inflammasome is considered an indicator of chronic inflammation in type 2 diabetes. Regular exercise training along with antioxidants supplements can control inflammation. The aim of the present study was to investigate the effect of training with different intensity and alpha lipoic acid supplement on pancreatic NLRP-3 expression in diabetic rats.
 Methods: In this experimental study, 35 Wistar rats were randomly divided into seven groups: control, diabetic, diabetic+ supplement (S), diabetic+ high intensity training (HIT), diabetic+ moderate intensity training (MIT), diabetic+ high intensity training + supplementation (HIT+S), diabetic+ moderate intensity training + supplementation (MIT+S). The HIT and MIT protocols were performed five days a week for six weeks. HIIT included 10 bouts of four minutes running (at 85–90% of VO2max) and MIT, 13 bouts of four minutes running (at 65–70% of VO2max). Alpha lipoic acid was administered orally at a dose of 20 mg/kg was given to rats once a day by gavage. Blood and tissue sample was taken 24 h after last exercise session. Real-time PCR method for the relative expression of NLRP-3 gene expression was used.
 Results: Expression of LRP-3 gene in pancreatic tissue was increased significantly in the diabetic group compared to control group (p=0.0001). In addition, S (P = 0.021), MIT (P = 0.009), HIT (P = 0.001), MIT+S (P = 0.003) and HIT+S (P = 0.0001), significantly reduced the NLRP-3 levels in pancreatic tissue compared to the diabetic group.
 Conclusion: Type 2 diabetes is associated with increased mRNA NLRP-3 in pancreatic tissue. MIT and HIT plus alpha lipoic acid can be considered as an effective intervention in decreasing pancreatic mRNA NLRP-3 in diabetic rats.

GSK-3β activates NF-κB to aggravate caerulein-induced early acute pancreatitis in mice

BackgroundAcute pancreatitis is a life-threatening disease which causes considerable morbidity and mortality. However, no specific and effective treatments are currently available for this critical condition, which is mainly due to the insufficient understanding of the early cellular events in the initial phase of acute pancreatitis. Previous researchers have reported that two independent events, intra-acinar trypsinogen and NF-κB activation, are of equal importance in the early development of acute pancreatitis. GSK-3β, an essential molecule in multiple physiopathological processes including inflammation, is associated with the expression of the NF-κB pathway.MethodsWe investigated whether GSK-3β affected the expression of cytokines produced by intra-acinar cells and aimed to determine the probable regulatory mechanism by using single allele GSK-3β-deficient mice.ResultsOur data showed that IL-6 and TNF-α mRNA expression in pancreatic tissue and serum IL-6 and TNF-α were significantly decreased. Meanwhile, pancreatic phospho-NF-κB p65 (ser536) protein expression in GSK-3β+/− mice was lower than that in wild type (WT) mice.ConclusionsGSK-3β may activate intra-acinar NF-κB signaling to promote the production of proinflammatory cytokines, which then induces the recruitment of inflammatory cells and activation of the cytokine cascade, further promoting local and systemic inflammation and ultimately aggravating acute pancreatitis. These findings strongly indicate that GSK-3β may be a potential treatment target for acute pancreatitis.

Pre-inhalation of hydrogen-rich gases protect against caerulein-induced mouse acute pancreatitis while enhance the pancreatic Hsp60 protein expression

BackgroundAcute pancreatitis (AP) lacks targeted prevention and treatment measures. Some key points in the pathogenesis of AP remain unclear, such as early activation of pancreatic enzymes. Several recent reports have shown the protective effect of hydrogen on several AP animal models, and the mechanism is related to antioxidant activity. Heat shock protein 60 (Hsp60) is known to accompany pancreatic enzymes synthesis and secretion pathway of in pancreatic acinar cells, while role of hsp60 in AP remains a topic. Aim of this study was to investigate effect of hydrogen pretreatment on AP and the mechanisms, focusing on pancreatic oxidative stress and Hsp60 expression.Methods80 mice were randomly assigned into four groups: HAP group, AP group, HNS group, and NS group and each group were set 3 observation time point as 1 h, 3 h and 5 h (n = 6–8). Mouse AP model was induced by intraperitoneal injection of 50 μg/kg caerulein per hour for 6 injections both in AP and HAP groups, and mice in NS group and HNS group given normal saline (NS) injections at the same way as control respectively. Mice in HAP group and HNS group were treated with hydrogen-rich gases inhalation for 3 days before the first injection of caerulein or saline, while mice in AP group and NS group in normal air condition. Histopathology of pancreatic tissue, plasma amylase and lipase, plasma IL-1 and IL-6, pancreatic glutathione (GSH) and malondialdehyde (MDA), and Hsp60 mRNA and protein expression were investigated. Comparisons were made by one-way analysis of variance.ResultsThe pancreatic pathological changes, plasma amylase and lipase activity, and the increase of plasma IL-1 and IL-6 levels in AP mice were significantly improved by the hydrogen-rich gases pretreatment, Meanwhile, the pancreatic GSH content increased and the pancreatic MDA content decreased. And, the hydrogen-rich gases pretreatment improved the Hsp60 protein expression in pancreatic tissues of AP mice at 1 h and 5 h.ConclusionsPre-inhalation of hydrogen-rich gases have a good protective effect on AP mice, and the possible mechanisms of reduced oxidative stress and the early increased pancreatic Hsp60 protein deserve attention.

Naringenin and Vanillin Mitigate Cadmium-Induced Pancreatic Injury in Rats via Inhibition of JNK and p38 MAPK Pathways

Background: Cadmium can induce pancreatic injury via oxidative stress, inflammation and apoptosis. Naringenin (NGN) and vanillin (VLN) exert antioxidant, anti-inflammatory, and antiapoptotic effects. Objective: The likely ameliorative effects of NGN, VLN and their combination were studied in rats exposed to cadmium-induced pancreatic injury. Materials and Methods: Rats received NGN (50 mg/kg/day, p.o.), VLN (100 mg/ kg/day, p.o.), or NGN + VLN for 7 days and one injection of CdCl2 (2 mg/kg, i.p.) on the 6th day. Results: Cadmium significantly lowered serum amylase and insulin levels. Cadmium also caused significant increments of malondialdehyde, tumor necrosis factor-α, interleukin-1β, nuclear factor-κB p65, Bax/Bcl-2 ratio and phosphorylated c-Jun N-terminal kinase (p-JNK) and p38 mitogen-activated protein kinases (MAPKs) and significant decrements of reduced glutathione and catalase in the pancreas of rats received CdCl2. Additionally, CdCl2 caused marked histopathological necrosis and significantly increased caspase-3 expression in pancreatic tissue. The cadmium-induced biochemical, histopathological and immunohistochemical changes were significantly ameliorated by NGN, VLN and NGN + VLN. However, NGN + VLN caused more significant ameliorative effects than did NGN and VLN alone. Conclusion: NGN, VLN and NGN + VLN afforded significant protection of pancreas in rats exposed to cadmium insult through modulation of JNK and p38 MAPK pathways and inhibition of oxidative stress, inflammation and apoptosis.

Transplantation of human dental pulp stem cells in streptozotocin-induced diabetic rats.

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease caused by the destruction of pancreatic β-cells. Human dental pulp stem cells represent a promising source for cell-based therapies, owing to their easy, minimally invasive surgical access, and high proliferative capacity. It was reported that human dental pulp stem cells can differentiate into a pancreatic cell lineage in vitro; however, few studies have investigated their effects on diabetes. Our study aimed to investigate the therapeutic potential of intravenous and intrapancreatic transplantation of human dental pulp stem cells in a rat model of streptozotocin-induced type 1 diabetes. Forty Sprague Dawley male rats were randomly categorized into four groups: control, diabetic (STZ), intravenous treatment group (IV), and intrapancreatic treatment group (IP). Human dental pulp stem cells (1 × 106cells) or vehicle were injected into the pancreas or tail vein 7days after streptozotocin injection. Fasting blood glucose levels were monitored weekly. Glucose tolerance test, rat and human serum insulin and C-peptide, pancreas histology, and caspase-3, vascular endothelial growth factor, and Ki67 expression in pancreatic tissues were assessed 28days post-transplantation. We found that both IV and IP transplantation of human dental pulp stem cells reduced blood glucose and increased levels of rat and human serum insulin and C-peptide. The cells engrafted and survived in the streptozotocin-injured pancreas. Islet-like clusters and scattered human dental pulp stem cells expressing insulin were observed in the pancreas of diabetic rats with some difference in the distribution pattern between the two injection routes. RT-PCR analyses revealed the expression of the human-specific pancreatic β-cell genes neurogenin 3 (NGN3), paired box 4 (PAX4), glucose transporter2 (GLUT2), and insulin in the pancreatic tissues of both the IP and IV groups. In addition, the transplanted cells downregulated the expression of caspase-3 and upregulated the expression of vascular endothelial growth factor and Ki67, suggesting that the injected cells exerted pro-angiogenetic and antiapoptotic effects, and promoted endogenous β-cell replication. Our study is the first to show that human dental pulp stem cells can migrate and survive within streptozotocin-injured pancreas, and induce antidiabetic effects through the differentiation and replacement of lost β-cells and paracrine-mediated pancreatic regeneration. Thus, human dental pulp stem cells may have therapeutic potential to treat patients with long term T1DM.

Extracellular vesicles microRNA analysis in type 1 autoimmune pancreatitis: Increased expression of microRNA-21

BackgroundThe molecular basis of type 1 autoimmune pancreatitis (AIP) remains unclear. Recent attention on the role of extracellular vesicles microRNA (EV miRNA) in immune homeostasis has prompted us to perform an extensive miRNA screening of serum-derived EV in AIP. MethodsEV miRNA expression was analyzed using microarrays in AIP, chronic pancreatitis (CP), and healthy adult (HC) samples (n = 10 from each group). Differences in signals, > 3 or <1/3 times, represented significant differences in expression. Another cohort of AIP (n = 14), CP (n = 10), and HC (n = 10) samples of EV miRNA was analyzed using reverse-transcription polymerase chain reaction (RT-PCR). miRNA expression in pancreatic tissues was evaluated using in situ hybridization (ISH) in three additional subjects from each group. ResultsSignals of eight miRNAs (miR-659–3p, −27a-3p, −99a-5p, -21–5p, -205–5p, -100–5p, −29c-3p, and −125b-1-3p) were significantly higher, while those of two miRNAs (miR-4252 and -5004–5p) were significantly lower in AIP than in HC. EV miR-21–5p was significantly up-regulated in AIP than in HC (P = 0.035) and CP (P = 0.048). The number of miR-21–5p positive inflammatory cells was significantly elevated in AIP than in CP (P = 0.014). ConclusionsCirculating EVs exhibited altered miRNA expression patterns with elevated miR-21–5p in AIP when compared with those in HC and CP. miR-21–5p was highly expressed in pancreatic inflammatory cells in AIP. Our data suggests that miR-21–5p may be involved in the regulation of effector pathways in the pathophysiology of AIP, thus differentiating AIP from CP.

PDAC-ANN: an artificial neural network to predict pancreatic ductal adenocarcinoma based on gene expression

BackgroundAlthough the pancreatic ductal adenocarcinoma (PDAC) presents high mortality and metastatic potential, there is a lack of effective therapies and a low survival rate for this disease. This PDAC scenario urges new strategies for diagnosis, drug targets, and treatment.MethodsWe performed a gene expression microarray meta-analysis of the tumor against normal tissues in order to identify differentially expressed genes (DEG) shared among all datasets, named core-genes (CG). We confirmed the CG protein expression in pancreatic tissue through The Human Protein Atlas. It was selected five genes with the highest area under the curve (AUC) among these proteins with expression confirmed in the tumor group to train an artificial neural network (ANN) to classify samples.ResultsThis microarray included 461 tumor and 187 normal samples. We identified a CG composed of 40 genes, 39 upregulated, and one downregulated. The upregulated CG included proteins and extracellular matrix receptors linked to actin cytoskeleton reorganization. With the Human Protein Atlas, we verified that fourteen genes of the CG are translated, with high or medium expression in most of the pancreatic tumor samples. To train our ANN, we selected the best genes (AHNAK2, KRT19, LAMB3, LAMC2, and S100P) to classify the samples based on AUC using mRNA expression. The network classified tumor samples with an f1-score of 0.83 for the normal samples and 0.88 for the PDAC samples, with an average of 0.86. The PDAC-ANN could classify the test samples with a sensitivity of 87.6 and specificity of 83.1.ConclusionThe gene expression meta-analysis and confirmation of the protein expression allow us to select five genes highly expressed PDAC samples. We could build a python script to classify the samples based on RNA expression. This software can be useful in the PDAC diagnosis.

Inflammatory biomarkers in the blood and pancreatic tissue of organ donors that predict human islet isolation success and function

ABSTRACTThe pancreas of brain-dead donors is the primary source of islets for transplantation. However, brain death mediates systemic inflammation, which may affect the quantity and quality of isolated islets. Our aim was to identify inflammatory biomarkers in donor blood and/or pancreatic tissue capable of predicting islet isolation success. Blood samples were collected from 21 pancreas donors and 14 healthy volunteers. Pancreatic tissue samples were also collected from the corresponding donor during organ procurement. Six serum cytokines were measured by a fluorescent bead-based immunoassay, and the expression of fifteen inflammatory target genes was quantified by quantitative reverse transcription polymerase chain reaction (RT-qPCR). There was no correlation between serum inflammatory cytokines and mRNA expression of the corresponding genes in peripheral blood mononuclear cells (PBMCs) or pancreatic tissue. The IL6 expression in pancreatic tissue correlated negatively with post-isolation islet yield. Islets isolated from donors highly expressing IFNG in PBMCs and MAC1 in pancreatic tissue functioned poorly in vivo when transplanted in diabetic NODscid mice. Furthermore, the increased MAC1 in pancreatic tissue was positively correlated with donor hospitalization time. Brain death duration positively correlated with higher expression of IL1B in PBMCs and TNF in both PBMCs and pancreatic tissue but failed to show a significant correlation with islet yield and in vivo function. The study indicates that the increased inflammatory genes in donor pancreatic tissues may be considered as biomarkers associated with poor islet isolation outcome.

Biochanin A improves insulin sensitivity and controls hyperglycemia in type 2 diabetes

Biochanin A (5,7-Dihydroxy-4′-methoxyisoflavone) is an O-methylated isoflavone known for its anti-inflammatory, lipid lowering and anti-cancer activity. The current study was designed to find out antidiabetic efficacy of Biochanin A in type 2 diabetes in rats.Induction of type 2 diabetes mellitus in experimental animals was carried out by manipulation of diet using high fat diet for fourteen days and then administration of streptozotocin at low dose of 35 mg/kg, i.p. The diabetic animals were treated with 10, 20 and 40 mg/kg of Biochanin A for 28 days. The effect of Biochanin A treatment in diabetic animals was evaluated by measuring changes in body weight, biochemical parameters, insulin sensitivity index, Homeostatic model assessment-Insulin resistance (HOMA-IR), oral glucose tolerance test, glycohaemoglobin and hepatic glycogen level. Changes in histopathological characteristics of pancreatic tissue were also evaluated after treatment with Biochanin A. Immunohistochemical analysis of pancreatic tissue was carried out for the expression of SIRT1.The results showed that the selected doses of (10, 20 and 40 mg/kg) Biochanin A significantly decreased blood glucose (p < 0.001). The higher dose (40 mg/kg) of Biochanin A significantly reduced glucose tolerance (p < 0.001) in diabetic animals. Biochanin A treatment significantly reduced insulin resistance (p < 0.001) and improved inulin sensitivity (p < 0.01 for 10 mg/kg, 20 mg/kg, p < 0.001 for 40 mg/kg) at all selected dose levels. It also improved lipid profile significantly (p < 0.001) at lower, middle and higher dose level. Glycohaemoglobin formation was significantly decreased in diabetic animals (p < 0.001) after treatment with Biochanin A at all three dose levels. Liver glycogen level was also improved significantly after treatment with Biochanin A in diabetic animals at 20 mg/kg and 40 mg/kg dose level. Biochanin A at dose of 40 mg/kg increased SIRT1 expression in pancreatic tissue. In conclusion, Biochanin A has significant effect in type 2 diabetes mellitus which might be linked to its effects on SIRT1.

Formononetin Treatment in Type 2 Diabetic Rats Reduces Insulin Resistance and Hyperglycemia.

Type 2 diabetic mellitus is a multifactorial metabolic disorder affecting huge population around the world. This indicates that there is an urgent unmet need of cost effective, new treatment strategies for type 2 diabetes mellitus with no or less side effects. Phenolic compounds including isoflavones are known for their beneficial effect in metabolic disorders. The present work was intended to find out efficacy of formononetin, an isoflavone treatment in experimental model of type 2 diabetes. Type 2 diabetes mellitus was induced by feeding high fat diet for 2 weeks prior to streptozotocin administration in Sprague Dawley rats. Diabetic animals were treated with formononetin for 28 days at three dose level, i.e., 10, 20, and 40 mg/kg body weight orally. The effect of formononetin treatment on various parameters such as plasma glucose, glucose tolerance, insulin, HOMA-IR, lipid profile, hepatic glycogen content, glycohaemoglobin and SIRT1 expression in pancreatic tissue was measured. Histopathological changes in pancreatic tissue were also studied. Results of the study demonstrate that formononetin treatment reduces blood glucose level significantly (p < 0.001) at all the three dose level. It also improved glucose tolerance, insulin sensitivity and lipid profile along with reduction in glycohaemoglobin content in blood. Formononetin treatment also improved hepatic glycogen level profoundly in diabetic rats. Determination of SIRT1 expression in pancreatic tissue by immunohistochemical analysis showed that formononetin treatment increases the expression of SIRT1 in pancreatic tissue. Histopathological study showed that treatment with formononetin protects pancreatic beta cells from necro-degeneration and atrophic effect. It can be concluded that formononetin treatment reduces insulin resistance and attenuate hyperglycemia in type 2 diabetes which may be due to increasing expression of SIRT1 in pancreatic tissues.

Novel Use of Agent 11C-PHNO for Pet CT Imaging of Pancreatic Beta-Cell Mass

There is growing interest in developing a method to measure pancreatic beta-cell mass (BCM) so as to: 1) monitor progression of beta cell changes in type 1 and type 2 diabetes; 2) identify therapies to preserve, restore or regenerate beta cells; and 3) monitor viability of islets after transplantation therapy. Given that beta cells and neurons have functional similarities, we tested the hypothesis that receptors, transporters, or proteins essential for signal detection and propagation, may be common to both cell types. Hence, we assessed if Positron Emission Tomography (PET) imaging ligands originally developed for neuroimaging may provide a means for imaging BCM as well. Previous studies have shown higher uptake of 11C-(+)-4-propyl-9-hydroxynaphthoxazine (PHNO), a dopamine D2/D3 agonist radioligand (D3-preferring), in the pancreatic tissue of healthy controls vs. T1DM in humans. In this study, we sought to determine whether 11C-PHNO, would be useful for measuring BCM. Eleven subjects (6 with T1DM, 4 males and 2 females, age 30.8 ± 11.4 years, BMI 24.4 ± 2.0, and 5 matched healthy controls, 3 males and 2 females, age 32.2 ± 11.4 years, BMI 26.4 ± 2.1) underwent abdominal PET/CT imaging using 11C-PHNO. Regions-of-interest (ROIs) were applied to the pancreas, liver, spleen, and kidney and time-activity curves (TACs) were produced and converted to standard uptake value (SUV) units. The distribution of activity between the head, body, and tail of the pancreas was also assessed. We observed that there was a 41% reduction in 11C-PHNO SUV in T1DM patients as compared to the healthy control group (p = 0.026). Moreover, there was a positive correlation between plasma c-peptide concentrations and 11C-PHNO SUV in the pancreas (r = 0.78), although this relationship did not reach statistical significance (p = 0.1), most likely due to the small sample size. These data suggest that dopamine D3 expression in pancreatic tissue might serve as a useful biomarker for assessing beta cell mass in diabetic humans. Disclosure E. Sanchez Rangel: None. J. Bini: None. N.B. Nabulsi: None. Y. Huang: None. K.C. Herold: None. R. Sherwin: Other Relationship; Self; QuintilesIMS, MannKind Corporation. Research Support; Self; Regeneron Pharmaceuticals, Inc.. Other Relationship; Self; ICON plc. R.E. Carson: Research Support; Self; Pfizer Inc., Bristol-Myers Squibb Company, Eli Lilly and Company, UCB, Inc., Siemens. G. Cline: None.

A proteome analysis of pig pancreatic islets and exocrine tissue by liquid chromatography with tandem mass spectrometry

ABSTRACTLiquid chromatography with tandem mass spectrometry (LC-MS/MS) is a proteome analysis method, and the shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolving power. In this study, we analyzed the protein expression in pancreatic tissue by LC-MS/MS. Islets isolated from porcine pancreata (purity ≥95%) and exocrine tissue (purity ≥99%) were used in this study. LC-MS/MS showed that 13 proteins were expressed in pancreatic islets only (Group I), 43 proteins were expressed in both islets and exocrine tissue (Group I&E), and 102 proteins were expressed in exocrine tissue only (Group E). Proteins involved in islet differentiation and cell proliferation were identified in Group I (e.g. CLUS, CMGA, MIF). In addition, various functional proteins (e.g. SCG2, TBA1A) were identified in islet by using the new method of ‘principal component analysis (PCA)’. However, the function of such proteins on islets remains unclear. EPCAM was identified in Group E. Group E was found to include proteins involved in clinical inflammatory diseases such as pancreatitis (e.g. CBPA1, CGL, CYTB, ISK1 and PA21B). Many of these identified proteins were reported less frequently in previous studies, and HS71B, NEC2, PRAF3 and SCG1 were newly detected in Group I while CPNS1, DPEP1, GANAB, GDIB, GGT1, HSPB1, ICTL, VILI, MUTA, NDKB, PTGR1, UCHL3, VAPB and VINC were newly detected in Group E. These results show that comprehensive expression analysis of proteins by LC-MS/MS is useful as a method to investigate new factors constructing cellular component, biological process, and molecular function.

Abstract 1442: Analysis of cis-eQTLs in normal and tumor-derived pancreatic tissues reveals functional insights, including for the 9q34.1 ABO pancreatic cancer risk locus

Abstract Objective: To elucidate the genetic architecture of gene expression in pancreatic tissues. Design: We performed expression quantitative trait locus (eQTL) and allele specific expression (ASE) analyses using RNA-sequence data and 1000 Genomes (1000G) imputed GWAS genotypes from 95 fresh frozen histologically normal pancreatic tissue samples. Data from 115 pancreatic tumor-derived tissue samples from The Cancer Genome Atlas (TCGA) was included for comparison. Results: We identified 38,615 cis-eQTLs (corresponding to 484 Genes) in histologically normal tissues and 39,713 cis-eQTL (corresponding to 237 Genes) in tumor tissues (FDR&amp;lt;0.1), with the strongest effects seen near transcriptional start sites (TSS). Approximately 23% and 42% of genes with significant cis-eQTLs (eGenes) appeared to be specific for tumor and normal derived tissues, respectively. Significant enrichment of cis-eQTL variants was noted in noncoding regulatory regions marked by modified histones, DNAse hypersensitivity and bound transcription factors, in particular for pancreatic tissues (1.53-3.12 fold, P≤0.0001), indicating tissue-specific functional relevance. A common pancreatic cancer risk locus on 9q34.2 in the ABO gene (rs687289) was associated with ABO expression in histologically normal (P=5.8x10-8) and tumor-derived (P=8.3x10-5) pancreatic tissues. The high linkage disequilibrium (LD) between this variant and the O blood group generating deletion variant in exon 6 of ABO suggested that nonsense-mediated decay (NMD) of the “O” mRNA could explain the eQTL. However, knock-down of crucial NMD regulators did not influence decay of the ABO “O” mRNA, indicating that a gene regulatory element influenced by pancreatic cancer risk alleles may underlie the eQTL. Conclusions: We have identified cis-eQTLs representing potential functional regulatory variants in the pancreas and generated a rich dataset for further studies on gene expression and regulation in pancreatic tissues. Citation Format: Laufey T. Amundadottir, Soren Lykke Andresen, Wenming Xiao, Jason Hoskins, Ashley Jermusyk, Lauren Rost, Irene Collins, Jinping Jia, Michael Mobaraki, Bin Zhu, Robert Kurtz, Hemang Parikh, Lei Song, Meredith Yeager, Torben Jensen, William Bamlet, Nilanjan Chatterjee, Brian Wolpin, Jill Smith, Sara Olson, Gloria Petersen, Jianxin Shi, Mingfeng Zhang. Analysis of cis-eQTLs in normal and tumor-derived pancreatic tissues reveals functional insights, including for the 9q34.1 ABO pancreatic cancer risk locus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1442. doi:10.1158/1538-7445.AM2017-1442

Heterogeneity of proliferative markers in pancreatic β-cells of patients with severe hypoglycemia following Roux-en-Y gastric bypass.

Severe postprandial hypoglycemia with neuroglycopenia is an increasingly recognized, debilitating complication of Roux-en-Y gastric bypass (RYGB) surgery. Increased secretion of insulin and incretin hormones is implicated in its pathogenesis. Histopathologic examination of pancreas has demonstrated increased islet size and/or nuclear diameter in post-RYGB patients who underwent pancreatectomy for severe refractory hypoglycemia with neuroglycopenia (RYGB+NG). We aimed to determine whether β-cell proliferation or apoptosis is altered in RYGB+NG. We performed an observational study to analyze markers of proliferation, apoptosis, cell cycle, and transcription factor expression in pancreatic tissue from affected RYGB+NG patients (n=12), normoglycemic patients undergoing pancreatic surgery for benign lesions (controls, n=6), and individuals with hypoglycemia due to insulinoma (n=52). Proliferative cell nuclear antigen (PCNA) expression was increased in insulin-positive cells in RYGB+NG patients (4.5-fold increase, p<0.001 vs. controls) and correlated with β-cell mass. Ki-67 immunoreactivity was low in both RYGB+NG and controls, but did not differ between groups. Phospho-histone H3 levels did not differ between RYGB+NG and controls. PCNA and Ki-67 were both significantly lower in both controls and RYGB+NG than insulinomas. Markers of apoptosis and cell cycle (M30, p27, and p21) did not differ between groups. PDX1 and menin exhibited similar expression patterns, while FOXO1 appeared to be more cytosolic in RYGB+NG. Markers of proliferation are heterogeneous in patients with severe post-RYGB hypoglycemia. Increased β-cell proliferation in some individuals may contribute to increased β-cell mass observed in severely affected patients.

Immunohistochemical assessment of glucagon-like peptide 1 receptor (GLP-1R) expression in the pancreas of patients with type 2 diabetes.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone which stimulates insulin release and inhibits glucagon secretion from the pancreas in a glucose-dependent manner. Incretin-based therapies, consisting of GLP-1 receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, are used for the treatment of type 2 diabetes (T2D). Immunohistochemical studies for GLP-1R expression have been hampered previously by the use of unspecific polyclonal antibodies. This study aimed to assess the expression levels of GLP-1R in a set of T2D donor samples obtained via nPOD. This study used a new monoclonal antibody to assess GLP-1R expression in pancreatic tissue from 23 patients with T2D, including 7 with a DPP-4 inhibitor and 1 with a history of GLP-1R agonist treatment. A software-based automated image analysis algorithm was used for quantitating intensities and area fractions of GLP-1R positive compartments. The highest intensity GLP-1R immunostaining was seen in beta-cells in islets (average signal intensity, 76.1 [±8.1]). GLP-1R/insulin double-labelled single cells or small clusters of cells were also frequently located within or in close vicinity of ductal epithelium in all samples and with the same GLP-1R immunostaining intensity as found in beta-cells in islets. In the exocrine pancreas a large proportion of acinar cells expressed GLP-1R with a 3-fold lower intensity of immunoreactivity as compared to beta-cells (average signal intensity 25.5 [±3,3]). Our studies did not unequivocally demonstrate GLP-1R immunoreactivity on normal-appearing ductal epithelium. Pancreatic intraepithelial neoplasia (PanINs; a form of non-invasive pancreatic ductular neoplasia) was seen in most samples, and a minority of these expressed low levels of GLP-1R. These data confirm the ubiquity of early stage PanIN lesions in patients with T2D and do not support the hypothesis that incretin-based therapies are associated with progression towards the more advanced stage PanIN lesions.