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E-GEOD-54774 - Expression data from mice on a high fat, high carbohydrate diet treated with exenatide

Status
Released on 1 June 2014, last updated on 31 July 2015
Organism
Mus musculus
Samples (12)
Array (1)
Protocols (7)
Description
The present study was constructed to confirm previous findings that mice on a high fat diet (HFD) treated by subcutaneous injection with exenatide (EXE) at 3µg/kg once daily for 6 weeks develop exocrine pancreatic injury (Rouse et al. 2014). The present study included 12 weeks of EXE exposure at multiple concentrations (3, 10, or 30 Œºg/kg) with multiple endpoints (histopathology evaluations, immunoassay for cytokines, immunostaining of the pancreas, serum chemistries and measurement of trypsin, amylase, and, lipase, and gene expression profiles). Time- and dose-dependent exocrine pancreatic injury was observed in mice associated with EXE exposure in a HFD environment. The time- and dose-dependent morphological changes identified in the pancreas involved acinar cell injury and death (autophagy, apoptosis, necrosis, and atrophy), cell adaptations (hypertrophy and hyperplasia), and cell survival (regeneration) accompanied with varying degrees of inflammatory response leading to secondary injury in pancreatic blood vessels, ducts, and adipose tissues. Gene expression profiles supported the presence of increased signaling for cell survival and altered lipid metabolism. The potential for EXE to cause acute or early chronic pancreatic injury was identified in a HFD environment. In human disease, the influence of pancreatitis risk factors or pre-existing chronic pancreatitis on this injury potential requires further investigation. Male C57BL/6 mice 6 to 8 weeks of age were purchased from Harlan Laboratories (Frederick, MD). Mice were housed individually in an environmentally controlled room (18C-21C, 40%-70% relative humidity) with a twelve-hour light/dark cycle. Mice were initially fed Certified Purina Rodent Chow #5002 (Ralston Purina Co., St. Louis, MO) providing 63% of calories through carbohydrates, 24% from protein, and 13% from fat. Per the experimental design, mice were subsequently placed on a Teklad Custom Research Diet, TD.06415 (Harlan, Frederick, MD), providing 45% of calories through fats (36% saturated, 47% monounsaturated, 17% polyunsaturated), 36% from carbohydrates, and 19% from protein. Water and food were available ad libitum. Thorough out the experiment all mice were weighed weekly and dosing adjusted accordingly for each mouse. A cohort of eighty 6 to 8 week-old male C57BL6 mice were received from Harlan Laboratories. The cohort represented experimental time point of 12 weeks. The cohort was further divided into 4 exenatide (EXE) treatment groups (0, 3, 10, or 30 ug/kg) of 20 mice each. Initially, all mice were placed on HFD for six weeks without additional treatment and were then maintained on HFD for an additional 12 weeks while receiving daily subcutaneous injections of saline or EXE (Creative Peptides, Shirley, NY; 3, 10, or 30 ug/kg). At the 12 week timepoint, twenty-four hours following their final dose, the mice from the treatment cohort were anesthetized with isoflurane and euthanized by exsanguination with collection of blood for serum harvest. The pancreas from 12 mice in each cohort was preserved in 10% formalin for histology processing. The pancreas from 5 mice in each cohort was placed in RNAlater (Life Technologies, Grand Island, NY) for subsequent RNA extraction and the pancreas from 3 mice in each cohort was immediately frozen in liquid nitrogen and then stored at -80C. Pancreas tissue was resected, preserved in RNALater, and stored at -80 C. After thawing, 2.5 mg of each sample were processed using miRNeasy Mini Kits (Cat # 217004) (Qiagen, Valencia, CA). Samples were homogenized in 700 uL of Qiazol Lysis Reagent (Qiagen), for 5 minutes at 50 hz in a TissueLyser LT (Qiagen), and processed using the automated purification of total RNA on a Qiacube (Qiagen), using the 'Purification of total RNA, including small RNAs, from animal tissues & cells (aqueous phase), version 2 (April 2010)' standard protocol, as described in the miRNeasy Mini Handbook (1073008 07/2012) & http://www.qiagen.com/QIAcube/Standard/ProtocolView.aspx?StandardProtocolID=847.Mean yield per 2.5 mg of pancreas tissue was approximately 35 ug, determined by NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). Average RIN values were approximately 5.4 +/- 1.4, as assayed by a 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA), using an Agilent RNA 6000 Nano Kit (cat # 5067-1511). This RIN mean was consistent with our previous experiences in RNA extraction from murine pancreatic tissue. The isolated RNA was labeled and gene expression analysis performed by Expression Analysis, Inc. (Durham, NC). The 3 samples with the highest RIN from each treatment group were used to probe treatment effect on gene expression. Total RNA samples (100 ng) were converted into cDNA using Ovation WGA FFPE System (NuGEN, Part No. 6200). The second strand cDNA is then purified with Agencourt RNAClean beads and followed by SPIA amplification. Amplified SPIA cDNA product is purified using Agencourt RNAClean beads and quantitated using a NanoDrop ND-8000 spectrophotometer. Target preparation was performed using 4 ug of amplified cDNA and the NuGEN FL-Ovation cDNA Biotin Module V2. Fragmented and biotin-labeled target was hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 microarrays. Raw gene expression data were first analyzed using ArrayTrack bioinformatics tool with robust multichip average algorithm and quantile normalization. Differentially expressed genes were defined by p < 0.05 and fold change > 1.3 in Welch t-test comparing treatment and control groups. Expression data were then imported to Ingenuity Pathway Analysis (IPA) tool for further analyses of pathway, biofunction, and toxicity using general and pancreas-specific knowledge bases.
Experiment type
transcription profiling by array 
Contacts
Citation
MIAME
PlatformsProtocolsVariablesProcessedRaw
Files
Investigation descriptionE-GEOD-54774.idf.txt
Sample and data relationshipE-GEOD-54774.sdrf.txt
Raw data (1)E-GEOD-54774.raw.1.zip
Processed data (1)E-GEOD-54774.processed.1.zip
Array designA-AFFY-45.adf.txt
Links