Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

RNA-seq of islet, FACS- sorted CD45+Ly6C-CD11B+CD11C+F4/80+ cells from C57bl6/J male mice that were treated with low dose streptozotocin (STZ) or acetate buffer

ABSTRACT: 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.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Mus musculus

SUBMITTER: Dominika Nackiewicz

PROVIDER: E-MTAB-7234 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Islet Macrophages Shift to a Reparative State following Pancreatic Beta-Cell Death and Are a Major Source of Islet Insulin-like Growth Factor-1.

Nackiewicz Dominika D Dan Meixia M Speck Madeleine M Chow Samuel Z SZ Chen Yi-Chun YC Pospisilik J Andrew JA Verchere C Bruce CB Ehses Jan A JA

iScience 20191214 1

Macrophages play a dynamic role in tissue repair following injury. Here we found that following streptozotocin (STZ)-induced beta-cell death, mouse islet macrophages had increased Igf1 expression, decreased proinflammatory cytokine expression, and transcriptome changes consistent with macrophages undergoing efferocytosis and having an enhanced state of metabolism. Macrophages were the major, if not sole, contributors to islet insulin-like growth factor-1 (IGF-1) production. Adoptive transfer exp ...[more]

PMID: 31962237

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Project description:Purpose. Patients with diabetic retinopathy may experience severe vision loss due to macular edema and neovascularization secondary to vascular abnormalities. However, before these abnormalities become apparent, there are functional deficits in contrast sensitivity, color perception, and dark adaptation. The goals of this study are to evaluate early changes (up to 3 months) in retinal gene expression, selected visual cycle proteins, and optokinetic tracking (OKT) in streptozotocin (STZ)-induced diabetic rats.Methods. Retinal gene expression in diabetic Long Evans rats was measured by whole genome microarray 7 days, 4 weeks, and three months after onset of hyperglycemia. Select gene and protein changes were probed by PCR and immunohistochemistry respectively, and OKT thresholds were measured using a virtual optokinetics system. Results. Microarray analysis showed that the most consistently affected molecular and cellular functions were cell-to-cell signaling and interaction, cell death, cellular growth and proliferation, molecular transport, and cellular movement. Further analysis revealed reduced expression of several genes encoding visual cycle proteins including lecithin:retinol acyltransferase (LRAT), retinal pigment epithelium (RPE)-specific protein 65kDa (RPE65) and RPE retinal G protein coupled receptor (RGR). Immunohistochemistry revealed a decrease in RPE65 in the RPE layer of diabetic rats. These molecular changes occurred simultaneously with a decrease in OKT thresholds by 4 weeks of diabetes. Conclusions. The data presented here are further evidence that inner retinal cells are affected by hyperglycemia prior to vasculopathy suggesting that glial and neuronal dysfunction may underlie some of the early visual deficits in diabetics. At each of the three timepoints (day 7, day 28, and day 84) one retina each from three diabetic rats were pooled for analysis on a single microarray chip. Three independent experiments were conducted for each group (n=9 animals/group). Each timepoint contained a hyperglycemic (STZ) and a control (buffer injection only) group. Additionally, on day 7 gene changes in the retina of rats which received a single injection of STZ, but did not develop hyperglycemia (STZ-non-c) were analyzed.

Dataset Information

RNA-seq of islet, FACS- sorted CD45+Ly6C-CD11B+CD11C+F4/80+ cells from C57bl6/J male mice that were treated with low dose streptozotocin (STZ) or acetate buffer

Publications

Islet Macrophages Shift to a Reparative State following Pancreatic Beta-Cell Death and Are a Major Source of Islet Insulin-like Growth Factor-1.

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