Expression data from pancreatic beta-cells obtained from type 2 diabetic subjects and non-diabetic controls
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Jeffrey Thorne
PROVIDER: E-GEOD-20966 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Islet transplantation exposes beta cells to mild hyperglycemia and to the abnormal environment of the transplant site. These conditions may affect beta cells and induce the expression of genes involved in beta cell damage. Gene expression profile of human beta cells exposed to mild hyperglycemia by transplantation into ICR-SCID mice was evaluated and compared with the gene profile of beta cells obtained from non-diabetic subjects. We found that the transplanted beta cells showed an unfolded protein response (UPR). There was upregulation of many genes of the IRE-1 pathway that provide protection against the deleterious effects of ER stress. Among them, increased expression of genes coding XBP-1; the chaperone proteins PDIA4, Bip, and Grp94; and the ER degradation proteins EDEM1 and EDEM2. ERdj4 and DNA-JC3 were also upregulated. JUK had downregulated expression. The PERK and ATF-6 arms of the ER stress response had many downregulated genes in transplanted islets. The PERK's substrates, EIF2A and NRF2, showed markedly reduced expression, as downregulated was the expression of CReP. Other downregulated genes included HERP2, IRS-2, CHOP and C/EBP-beta. In the transplanted beta cells there was significantly decreased expression of ATF-6, as well as the downstream gene products CHOP and HERP2. There was increased expression of HRD1, which exerts an antiapoptotic effect by degrading unfolded proteins. In conclusion human beta cells in a transplant site had UPR changes in gene expression that protect against the proapoptotic effects of unfolded proteins. Frozen sections were obtained from pancreases of non-diabetic subjects at surgery and from human islets transplanted into ICR-SCID mice for 4 weeks. β cell enriched samples were obtained by laser capture microdissection. RNA was extracted, amplified and subjected to microarray analysis.
2010-06-08 | E-GEOD-22097 | biostudies-arrayexpress
Project description:We demonstrate diverse roles of interferonâgamma (IFN-γ) in the induction and regulation of immune-mediated inflammation using a transfer model of autoimmune diabetes. The diabetogenic CD4+BDC2.5 (BDC) T cell clone upon transfer into NOD.scid mice induced destruction of islets of Langerhans leading to diabetes. Administration of a neutralizing antibody to IFN-γ (H22) resulted in long term protection (LTP) from diabetes, with inflammation but persistence of a significant, albeit decreased numbers of β-cells. BDC T cells were a mixture of cells expressing high, intermediate and low levels of the T cell receptor. Clonotype-low BDC T cells were required for LTP. Furthermore, islet infiltrating leukocytes in the LTP mice contained Foxp3+CD4 T cells. Islet inflammation in both diabetic and LTP mice was characterized by heavy infiltration of macrophages. Gene expression profiles indicated that macrophages in diabetic mice were M1-type, while LTP mice contained M2-differentiated. The LTP was abolished if mice were treated with either an antibody depleting CD4 T cells, or a neutralizing antibody to CTLA-4, in this case, only at a late stage. Neutralization of IL-10, TGF-β, GITR or CD25 had no effect. Transfer of only clonotype-high expressing BDC T cells induced diabetes but in contrast, H22 antibodies did not inhibit diabetes. While clonotype high T cells induced diabetes even when IFN-γ was neutralized, paradoxically, there was reduced inflammation and no diabetes if host myeloid cells lacked IFN-γ receptor. Hence, using monoclonal CD4 T cells, IFN-γ can have a wide diversity of roles, depending on the setting of the immune process. Experiment Overall Design: Pancreatic islets were laser-capture microdissected from mice injected with diabetogenic T cells. One cohort of mice also received injections with anti-interfereon gamma monoclonal antibody, which protected those mice from developing diabetes. RNA prepared from islets was amplified and analyzed by Affymetrix GeneChips. Each GeneChip was prepared from RNA pooled from 5 mice at each timepoint. GeneChips were prepared from RNA extracted at different days following injection of T cells. The following days were assayed day 0 (i.e., untreated), day 3 (for diabetic and protected islets), day 4 (diabetic and protected), day 5 (only for protected, as diabetic islets were too edematous to dissect), day 8 (diabetic and protected).
2008-11-13 | E-GEOD-12389 | biostudies-arrayexpress
Project description:We recently reported the scalable in vitro production of functional stem cell-derived β cells. Here we extend this approach to generate SC-β cells from Type 1 diabetic patients (T1D), a cell type that is destroyed during disease progression and has not been possible to extensively study. These cells express β cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice, and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of β cell stress. Using these assays, we find no major differences in T1D SC-β cells compared to SC-β cells derived from non-diabetic patients (ND). These results show that T1D SC-β cells can be used for the treatment of diabetes, drug screening, and the study of β cell biology. Differentiated cells were sorted and processed for RNA isolation using the MARIS protocol published previously (PMID: 24516164.) Human induced pluripotent stem cell (hiPSC) line were differentiated into SC-beta cells or dysfunctional, polyhormonal cells (PH). Four biological replicates were assessed with differentiation to both SC-beta and PH cells. Those data were normalized together with and compared to existing, previously published data from Hrvatin et al. (PMID: 24516164) and Pagliuca et al. (PMID: 25303535) from human islet-derived insulin+ cells, undifferentiated HUES8 hES cells, SC-beta cells derived from HUES8 and PH cells derived from HUES8 according to previously published protocols.
2016-04-01 | E-GEOD-70901 | biostudies-arrayexpress
Project description:Fetal and neonatal beta cells have poor glucose-induced insulin secretion and only gain robust glucose responsiveness several weeks after birth. This unresponsiveness may be due to a generalized immaturity of the metabolic pathways normally found in beta cells. Gene expression profile of neonatal (1 day old) and young adult (6 weeks old) Sprague-Dawley rat islets were evaluated and compared. Frozen sections were obtained from pancreases of neonatal (1 day old) and young adult (6 weeks old) Sprague-Dawley rats. The beta cell enriched cores of the islets were excised using laser capture microdissection. RNA was extracted, amplified and subjected to microarray analysis.
2011-02-22 | E-GEOD-24790 | biostudies-arrayexpress
Project description:Urinary exosomal miRNA profiling was conducted in urinary exosomes obtained from 8 healthy controls (C), 8 patients with type II diabetes (T2D) and 8 patients with type II diabetic nephropathy (DN) using Agilent´s miRNA microarrays.
2016-11-03 | E-MTAB-4166 | biostudies-arrayexpress
Project description:Background: In the diabetic heart the β-adrenergic response is altered partly by down-regulation of the β1-adrenoceptor, reducing its positive inotropic effect and up-regulation of the β3-adrenoceptor, increasing its negative inotropic effect. Statins have clinical benefits on morbidity and mortality in diabetic patients which are attributed to âpleiotropicâ effects. The objective of our study was to investigate the role of statin treatment on β-adrenergic dysfunction in diabetic rat cardiomyocytes. Methods: β-adrenergic responses were investigated in vivo (echocardiography) and ex vivo (left ventricular papillary muscles) in healthy and streptozotocin-induced diabetic rats, who were pre-treated or not by oral atorvastatin over 15 days (50 mg.kg-1.day-1). Micro-array analysis and immunoblotting were performed in left ventricular homogenates. Data are presented as mean percentage of baseline ± SD. Results: Atorvastatin restored the impaired positive inotropic effect of β-adrenergic stimulation in diabetic hearts compared with healthy hearts both in vivo and ex vivo but did not suppress the diastolic dysfunction of diabetes. Atorvastatin changed the RNA expression of 9 genes in the β-adrenergic pathway and corrected the protein expression of β1-adrenoceptor and β1/β3-adrenoceptor ratio, and multidrug resistance protein 4 (MRP4). Nitric oxide synthase (NOS) inhibition abolished the beneficial effects of atorvastatin on the β-adrenoceptor response. Conclusions: Atorvastatin restored the positive inotropic effect of the β-adrenoceptor stimulation in diabetic cardiomyopathy. This effect is mediated by multiple modifications in expression of proteins in the β-adrenergic signaling pathway, particularly through the NOS pathway.
2017-06-27 | E-MTAB-5594 | biostudies-arrayexpress
Project description:In the present study, we aimed to determine the genes involved in inflammatory process of diabetic nephropathy. ICAM-1+/+ and ICAM-1-/- mice aged 8 weeks were divided into four groups: 1) nondiabetic ICAM-1+/+ mice (ND-WT), 2) nondiabetic ICAM-1-/- mice (ND-KO), 3) streptozotocin (STZ)-induced diabetic ICAM-1+/+ mice (DM-WT), and 4) STZ-induced diabetic ICAM-1-/- mice (DM-KO). Three months after the induction of diabetes, total RNA was extracted from each specimen of renal cortex. We examined gene expression profiles of four groups. We identified 193 genes; the ratio of expression level of DM-WT was >2 or <0.5 of that of DM-KO. Of 193 genes, hierarchical clustering identified 33 genes that were significantly upregulated only in DM-WT but not remarkable in ND-WT and ND-KO. Functional annotation of these 33 genes revealed that the significant functions of them were related to the immune or inflammatory process: immune response, response to stimulus, defense response, immune effector process and antigen processing and presentation. These genes contained several inflammatory related genes, such as chemokine (C-X-C motif) ligand 10, chemokine (c-c motif) ligand 12, and chemokine (c-c motif) ligand 8. In this cluster, we focused on cholecystokinin (CCK) because CCK is one of the most up-regulated genes. Real-time RT-PCR revealed that CCK mRNA expression was significantly up-regulated in DM-WT compared with DM-KO. These results suggest that CCK may play a critical role in the progression of diabetic nephropathy by controlling inflammation in diabetic kidney. Male ICAM-1-/- mice (C57BL/6J background) were purchased from The Jackson Laboratory (Bar Harbor, ME). Male C57BL/6J mice (ICAM-1+/+) mice were used as controls. ICAM-1+/+ and ICAM-1-/- mice aged 8 weeks were divided into four groups (n = 5 each): 1) nondiabetic ICAM-1+/+ mice (ND-WT), 2) nondiabetic ICAM-1-/- mice (ND-KO), 3) streptozotocin (STZ)-induced diabetic ICAM-1+/+ mice (DM-WT), and 4) STZ-induced diabetic ICAM-1-/- mice (DM-KO). Mice in the diabetic groups received an intraperitoneal injection of STZ (Sigma Chemical, St.Louis, MO) at 200 mg/kg in citrate buffer (pH 4.5). Blood glucose levels were determined 7 days after STZ injection and only mice with blood glucose concentrations >16 mmol/L were used in the study. Nondiabetic ICAM-1+/+ and ICAM-1-/- mice received citrate buffer injections only. All mice had free access to standard diet and tap water. All animal procedures were performed according to the Guidelines for Animal Experiments at Okayama University Medical School, Japanese Government Animal Protection and Management Law (no. 105), and Japanese Government Notification on Feeding and Safekeeping of Animals (no. 6). Three months after the induction of diabetes, all mice were killed, and the kidneys were harvested. Total RNA was extracted from each specimen of renal cortex using standard protocol from RNeasy midi kit (Qiagen, Valencia, CA) at 3 months. The concentration and the quality of the total RNA sample were assessed by Agilent Bioanalyzer. Biotin-labeled target cRNA was prepared using First and Second strand cDNA Synthesis Kit (Amersham Biosciences, No. 320000) and In Vitro Transcription Kit (Amersham Biosciences, No.320001). Incubate total RNA (5 μg), diluted bacterial mRNA spike controls and T7 oligo (dT) primer for 10 min at 70 C. Add first-strand reaction components and incubate 1 hour at 42C. Add the first-strand cDNA product to the second-strand reaction components and incubate for 2 hours at 16 C. Double-strand cDNA was purified using QIAquick purification kit (Qiagen), eluted twice, each time with 30μl of nuclease-free water then dried in a SpeedVac concentrator. Prepare IVT mix of biotinylated UTP, ribonucleotides, and the 10x T7 enzyme mix and add to the resuspended cDNA. Incubate at 37C for 14 hours. Purify cRNA using RNeasy Mini Kit (Qiagen) and elute the cRNA twice, each time with 50 μl nuclease-free water. Measure the absorbance at 260 nm and 280 nm to determine the ratio (A260:A280=2). In 25 μl total volume, add 10 μg of cRNA to 5 μl of 5x fragmentation buffer and incubate at 94 C for 20 min. Bring 10 μg of fragmented cRNA, 78 μl of hybridization buffer component A, and 130 μl of hybridization buffer component B to a final volume of 260 μl with water. Incubate at 90C for 5 min and immediately chill on ice for 5 min. Slowly inject 250 μl of hybridization reaction mixture into array input port and seal ports with sealing strips. Set the shaker speed to 300 rpm and incubate slides for 18 hours at 37C in an Innove 4080 shaker. Remove the Flex Chamber using the hybridization removal tool. Then, place the bioarrays into the bioarray rack while it sits inside the medium reagent reservoir containing 0.75xTNT. Transfer the bioarray rack to the large reagent reservoir containing preheated 0.75xTNT, and incubate at 46 C for exactly 1 hour. Fill each slot of the small reagent reservoir with 3.4 ml of Cy5-Streptavidin working solution. Transfer the bioarray rack from the large reagent reservoir in to the small reagent reservoir and incubate bioarrays at RT for 30 min. Wash the bioarrays four times with 1xTNT at RT for 5 min. Rinse the bioarrays in 0.1xSSC/0.05%Tween by moving rack up and down 5 times in 5 seconds. Immediately follow the rinse with centrifugation to dry bioarrays, and store dried bioarrays in the dark. We scanned bioarrays with Axon GenePix 4000B and analyzed with CodeLink Expression Analysis software version 2.3.2. The 10,000 spot intensities on the scanned microarray image were normalized to a median value of 1 and data were exported for analysis with CodeLink Expression Analysis software version 2.3.2
2014-10-08 | E-GEOD-21390 | biostudies-arrayexpress
Project description:Diabetic islets
2009-06-30 | E-MEXP-1878 | biostudies-arrayexpress
Project description:Objective: To develop a reliable, reproducible, and sensitive method for investigating gene-expression profiles from individual human oocytes. Setting: University medical center and university microarray laboratory. Patient(s): Couples undergoing intracytoplasmic sperm injection treatment were asked to donate their immature oocytes for research, and written informed consent was obtained in all cases. Seventy-nine human oocytes were used in total. Intervention(s): None. Main Outcome Measure(s): Amplification efficiency and microarray profiles. Result(s): Two of the five protocols (WT-Ovation One-Direct and Arcturus RiboAMp HS Plus) resulted in sufficient yields and high success rates and were further validated for their performance in obtaining reliable, reproducible, and sensitive expression profiles from individual human oocytes. Evaluation of these two protocols demonstrated that they both displayed low technical variation and produced highly reproducible profiles (r R 0.95). One of them identified significantly more transcripts but also had a higher number of false discoveries. Conclusion(s): Two protocols generated ample amounts of mRNA for (quantitative) polymerase chain reaction, microarray, and sequencing techniques. Further validation using a design that discriminates between biological and technical variation showed that both protocols can be used for gene-expression profiling of individual human oocytes. Design: Five commercially available protocols were investigated for their efficiency to amplify messenger RNA (mRNA) from 54 single human oocytes. Protocols resulting in sufficient yields were further validated using microarray technology. For the validation, mRNA was isolated from 25 human oocytes. To eliminate biological variation, RNA from 13 human oocytes was pooled together and split into 12 identical samples for further mRNA amplification. From 12 oocytes, mRNA was individually isolated.
2015-12-31 | E-GEOD-48141 | biostudies-arrayexpress
Project description:Podocytes form filtration barrier through foot process around glomerualar basement membrane and selectively permit permeability of molecular smaller than albumin. Diabetes can cause podocyte pathological changes leading to high urine albumin level. Diabetic mouse model OVE26 has extremly high urine albumin and previously studies indicated its podocyte damaged. Here we try to find the key genes change in OVE26 diabetic mouse model podocyte by microarray assay while normal FVB mouse podocyte set as control. Podocyte eGFP transgenic mice were made on FVB background and crossbred to OVE26 diabetic model. Glomeruli isolated from OVE-GFP mice were digested by trypsin into signal cell. Podocytes with GFP were sorting out by FACS.
2012-03-02 | E-GEOD-36209 | biostudies-arrayexpress