Microarray analysis of islets of Langerhans from 3 week-old NOD, NOR and C57Bl/6 mice
Ontology highlight
ABSTRACT: The aim of this study was to investigate which intrinsic differences are present in the islets of Langerhans from diabetes-prone non-obese diabetic (NOD) mice before the onset of insulitis.
Project description:Zfp92, a repressive KRAB domain-containing zinc-finger protein, was identified by Gene Co-expression Network analysis to be an interesting candidate gene involved in endocrine specification and maturation. We examined the role of Zfp92, a KRAB-ZFP that is highly expressed in pancreatic islets of adult mice, by analyzing global Zfp92 knockout (KO) mice. Adult Zfp92 KO animals exhibited only mild changes in glucose homeostasis and no change in islet structure, although, male KO mice exhibited decreased growth, and female KO mice exhibited increased body fat accumulation on a high fat diet. We found that Zfp92 regulates a subset of transposable elements as well as Mafb, a transcription factor involved in islet development.
Project description:Nkx6.1 target genes were identified in mature pancreatic islets by comparing gene expression in conditional Nkx6.1-ablated islets versus control islets using microarray analysis. Nkx6.1 was conditionally ablated in mature pancreatic islets by recombination of a Nkx6.1-flox allele using the tamoxifen-inducible Pdx1-CreERTM allele (Gu et al 2002). Mice were injected with 2 mg/25 g tamoxifen in corn oil four times between 4 and 6 weeks of age. Islets were isolated after the final tamoxifen injection. Total RNA was isolated and pooled from pancreata of 6 week old Nkx6.1fl/-;Pdx1-CreERTM (mutant) versus Nkx6.1fl/+;Pdx1-CreERTM (control) littermates for 3 biological replicates.
Project description:Beta-cells produce hybrid insulin peptides (HIPs) by linking insulin fragments to other peptides through peptide bonds. HIPs have unique amino acid sequences and are targeted by autoreactive T cells in type 1 diabetes (T1D). Individuals with recent-onset T1D have significantly higher levels of HIP-reactive T cells in their blood compared to non-diabetic control subjects. HIP-reactive T cells have also been found in the residual pancreatic islets of deceased T1D organ donors. In non-obese diabetic (NOD) mice, a major T1D animal model, several CD4 T cell clones that trigger diabetes have been shown to target HIPs. Through mass spectrometry, a subgroup of HIPs containing N-terminal amine groups of various peptides linked to aspartic acid residues of insulin C-peptide has been detected in NOD islets. Our research reveals that these HIPs form spontaneously in beta-cells via an aspartic anhydride intermediate mechanism. This process leads to the creation of a regular HIP with a standard peptide bond and a HIP-isomer (isoHIP) with an isopeptide bond linked to the carboxylic acid side-chain of the aspartic acid residue. Our mass spectrometric analyses confirmed the presence of both HIP isomers in murine islets, thereby validating the occurrence of this new reaction mechanism in beta-cells. The spontaneous formation of neoepitopes through the development of new peptide bonds within cells may contribute to the pathogenesis of T1D and other autoimmune diseases.
Project description:To determine the role of Ascl1 in beta cell development, function, and metabolic stress response, we generated beta cell specific Ascl1 knockout mice and assessed their glucose homeostasis, islet morphology, and gene expression after feeding a normal diet, a high fat diet (HFD) for 12 weeks, or on a background of Abcc8 (KATP channel subunit) knockout mice. For the RNA-seq analysis, islets from male Ascl1betaKO and littermate control mice (N = 4 for each genotype and condition) were collected from three different conditions: 1) normal diet fed 2) HFD fed for 12 weeks, 3) on a background of homozygous Abcc8 allele.
Project description:In the context of T1 Diabetes, pro-inflammatory cytokines IL-1β and IFN-γ are known to contribute to β-cell apoptosis; The measurement of mRNA expression following β-cell exposure to these cytokines gives a picture of the changes in gene expression characterizing the path to β-cell dysfunction and death. INS1 cell lines were cultured in medium with or without IL-1β and IFN-γ. The samples were collected at various time points for profiling with Affymetrix Rat ST arrays. These experiments were performed on two separate occasions.
Project description:The tumor microenvironment plays a critical role in cancer progression, but the precise mechanisms by which stromal cells influence the tumor epithelium are poorly understood. The signaling adapter p62 has been implicated as a positive regulator of epithelial tumorigenesis; however, its role in the stroma is unknown. We show here that p62 levels are reduced in the stroma of several tumors. Also, orthotopic and organotypic studies demonstrate that the loss of p62 in the tumor microenvironment or stromal fibroblasts resulted in increased tumorigenesis of epithelial prostate cancer cells. The mechanism involves the regulation of cellular redox through an mTORC1/c-Myc pathway of stromal glucose and amino acid metabolism. Inhibition of the pathway by p62 deficiency results in increased stromal IL-6 production, which is required for tumor promotion in the epithelial compartment. Thus, p62 is an anti-inflammatory tumor suppressor that acts through modulation of metabolism in the tumor stroma. C57BL6 syngeneic TRAMP-C2Re3 cells (5×10^4) were injected orthotopically into the prostate of WT (n=6) or p62 KO (n=6) mice (C57BL6 background) and tumor growth was assessed, followed by transcriptome profiling of the orthotopic tumors (n=12).
Project description:C57BLKS/J mice are susceptible to diabetes, because of islet dysfunction, whereas C57BL6/J mice are not. Differences in gene expression between the two strains may account for this sensitivity. Furthermore these differences may only be evident in the hyperstimulated (diabetic or hyperglycemic) state. To this end profiling islets from these two strains cultured in both low and high glucose may reveal the underlying mechanism. Keywords: Mouse strain comparison under different culture conditions In the study presented here, pancreatic islets from 20 mice grown in low and high glucose conditions were assayed for differences in gene expression. (five C57BLKS/J low glucose, four C57BLKS/J high glucose, six C57BL6/J low glucose, five C57BL6/J high glucose). Technical replicates are achieved by labeling each sample with both Cy3 and Cy5, and combining the values for each hybridization.
Project description:The experiment was designed to obtain a broader unbiased view of the changes in islet macrophages following low dose STZ challenge. Mice were purchased from Jackson Laboratory (Bar Harbor, ME). 16-20-week-old C57BL/6J males were given 30 mg/kg STZ or acetate buffer (control) i.p. (intraperitoneal injection) for 5 consecutive days. Following the first STZ or buffer injection mice were sacrificed on day 14 and islets were isolated by collagenase digestion. Freshly isolated islets were dispersed in 0.02% Trypsin-EDTA for 3 minutes followed by up to 1 minute of pipetting under a stereomicroscope to obtain a single cell solution. Islet media was added to stop the reaction. Islets from 10 mice were pooled per sample (N). Dispersed islets were washed with FACS buffer (1% heat inactivated FBS, 1 mM EDTA, 11 mM glucose in PBS). Cells were kept on ice and pre-incubated with Fc Block (1:100) for 5 minutes, followed by 30 min incubation with CD45-eFluor 450 (1:250; clone 30-F11), Ly-6C-APC (1:1,200; clone HK1.4), CD11b-PE (1:1,200; clone M1/700, F4/80-FITC (1:150; clone BM8), CD11c-PECy7 (1:150; clone N418), and the viability dye 7AAD (1:2,000). Unstained, single stains, and fluorescence minus one controls were used for setting gates and compensation. Viable, single CD45+Ly6c-Cd11b+Cd11c+F4/80+ cells were sorted using a BD FACS Aria IIu directly into lysis buffer, and the RNeasy Plus Micro Kit from Qiagen was used to isolate total RNA. Total RNA quality control quantification was performed using an Agilent 2100 Bioanalyzer. All RNA samples had an RNA integrity number (RIN) ≥9.1. The NeoPrep Library Prep System from Ilumina was used for library preparation followed by sequencing using standard Illumina methods and Ilumina NextSeq500.
Project description:Previous studies have shown that a considerable proportion of the Arabidopsis genome cycles under circadian and/or diurnal conditions (Edwards et al., 2006 PMID: 16473970, Blaesing et al., 2005 PMID 16299223). It is likely that the correct phasing of gene expression plays an important role in improving the growth of the model plant (Dodd et al., 2005 PMID: 16040710). A key question is whether similar regulation is occurring in other higher plant species. In this study we measure the diurnal expression pattern of genes in the T89 clone of the hybrid aspen (Populus tremula L. x P. tremuloides Michx.) using the Affymetrix Poplar array. Samples (leaf blades) were taken at 4h intervals over the course of 2 diurnal cycles (18h Light: 6h Dark). Time-points were labelled relative to dawn (time 0) on the first day of sampling. T89 was selected as it has routinely been used for genetic modification since 1992 (Nilsson et al.) and is the genetic background for a large number of transgenic trees created in order to investigate the function of aspen genes, providing potential for genetic manipulations during our further studies. We also measured the diurnal expression pattern of genes in trees with a compromised circadian clock. Trees with less expressed LATE ELONGATED HYPOCOTYL 1 (LHY1) and LHY2 by RNA interference (lhy) line that was created in T89 clone of the aspen hybrid (Populus tremula L. x P. tremuloides Michx.) using the Affymetrix Poplar array. Samples (leaf blades) were taken at 4h intervals over the course of 2 diurnal cycles (18h Light: 6h Dark) as described above. Thus, time-points are labelled relative to dawn (time 0) on the first day of sampling. The data obtained from using a RNAi line in central Clock components complements the study of wild type T89, since they were grown and sampled at the same time under the same conditions.