Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomic characterization of the diabetic retinal response to chronic insulin treatment

ABSTRACT: The aim of this study was to identify alterations associated with diabetes-induced functional dysregulation of the retina and the effects of chronic insulin therapy. Transcriptional profiling through microarray analysis was analyzed to find mRNA targets of interest in diabetic samples, as well as in the insulin treated group. Verification of targets shown to be unrecovered in the insulin group was validated. Diabetes was induced in Sprague-Dawley male rats (Charles River Laboratories, Wilmington, MA) by intraperitoneal injection of 65 mg/kg streptozotocin (STZ)(Sigma-Aldrich, St. Louis, MO) in 10mM sodium citrate pH 4.5 vehicle. Control rats were injected with an equal dose of vehicle only. Rats had free access to food and water, and were maintained on a 12 hour light/dark cycle. Blood glucose level and body weight were measured 6 days post-STZ or vehicle injection, and biweekly throughout the experiment. Only rats with blood glucose levels >250 mg/dL at the time of the original test and throughout the experiment were included in the diabetic groups. The insulin treatment group received one 26mg subcutaneous pellet (LinShin Canada, Scarborough, Canada) delivered via trocar 6 weeks post-STZ injection. An additional 26 mg implant was introduced when body weight exceeded 300 g or when midday non-fasting blood glucose exceeded 250 mg/dL. At the time of retina harvest, rats were given a lethal dose of pentobarbital, 100 mg/kg, (Ovation Pharmaceuticals Inc., Deerfield, IL) by intraperitoneal injection and sacrificed by decapitation. Retinas were rapidly excised snap- frozen in liquid nitrogen for subsequent experimentation.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Robert Brucklacher

PROVIDER: E-GEOD-24423 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Chronic insulin treatment of diabetes does not fully normalize alterations in the retinal transcriptome.

Bixler Georgina V GV Vanguilder Heather D HD Brucklacher Robert M RM Kimball Scot R SR Bronson Sarah K SK Freeman Willard M WM

BMC medical genomics 20110515

<h4>Background</h4>Diabetic retinopathy (DR) is a leading cause of blindness in working age adults. Approximately 95% of patients with Type 1 diabetes develop some degree of retinopathy within 25 years of diagnosis despite normalization of blood glucose by insulin therapy. The goal of this study was to identify molecular changes in the rodent retina induced by diabetes that are not normalized by insulin replacement and restoration of euglycemia.<h4>Methods</h4>The retina transcriptome (22,523 ge ...[more]

PMID: 21575160

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Project description:Background and Aims: It is well demonstrated that in the beta cell population of the pancreas there is a dynamic turnover, which results from the net balance of several processes; beta cell replication, apoptosis and neogenesis. These processes have been studied in partial pancreatectomy and glucagon-like peptide 1 treated animals, where an increase in pancreas regeneration has been observed. Similarly, sodium tungstate, which decreases hyperglycemia in several animal models of diabetes, promotes a rise in the beta cell mass of nSTZ and STZ animals. However, the molecular mechanisms underlying this pancreas regeneration remain unknown. Therefore the objective of this study is to identify which genes are up or down regulated in the increase of the beta cell population of STZ rats treated with sodium tungstate. Materials and methods: Adult male Wistar (225-250 g) rats were kept under a constant 12-hour light-dark cycle and rats were kept under a constant 12-hour light-dark cycle and were allowed to eat and drink freely. Diabetes was induced by a single i.p. injection of streptozotocin (STZ) (70 mg/Kg body weight) in 0.9% NaCl with 100 mmol/L sodium citrate buffer (pH 4.5). Diabetes was confirmed by determination of its hyperglycaemia (>500mg/dL [Reflotron, Roche Diagnostic]). Healthy rats received an i.p. injection of the vehicle. Treatment started 7 days after the STZ or vehicle injection. Diabetic and healthy rats were divided into two groups. In the first (untreated), rats received deionized drinking water; in the second (treated) group, they were given a solution of sodium tungstate. During the first week of treatment, the rats received a solution of 0.7 mg/mL and in the next 4-5 weeks, the concentration was increased to 2 mg/mL. At the end of the experiment, the animals were sacrificed and pancreatic RNA isolated. Three chips (Affymetrix RAE-230A) were hybridized for each of the four experimental groups (untreated and treated healthy rats and untreated and treated diabetic rats). The raw intensity data obtained from the microarrays was normalized and summarized using the Bioconductor package RMA. Keywords = pancreas regeneration Keywords = STZ Keywords = diabetes Keywords = tungstate Keywords = insulin-like agents Keywords = beta cell plasticity Keywords = neogenesis Keywords: ordered

Project description:Experiments were conducted using 13-wk-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME). Mice were housed individually with free access to water and chow (TD.10112; Harlan Laboratories, Indianapolis, IN), with a 12:12-h light-dark cycle and temperature and humidity control. Mice were acclimated for 1 wk prior to the beginning of the experiment. The protocol was approved by the Mayo Clinic Institutional Animal Care and Use Committee. Following a 6-h fast, mice were given intraperitoneal injections of STZ (125 mg/kg; in sodium acetate buffer, pH = 4.5) (67). Injections were repeated on the following day. Control animals received intraperitoneal injection of vehicle. Only mice that displayed blood glucose ?300 mg/dl and an increase in blood ketones (both values by Precision Xtra glucometer; Abbott Laboratories, Abbott Park, IL), hyperphagia, and polyuria and were positive for urine glucose presence via dipstick (Uristix, Bayer, Pittsburgh, PA) on day 7 after the first STZ dose were included in the experiment. Animals that were positive for STZ diabetes received LinBit subcutaneous insulin implant (LinShin Canada, Toronto, ON, Canada) (79) under pentobarbital sodium anesthesia (Nebutal, 40 mg/kg of body wt) according to the manufacturer's protocol. Each animal received two subcutaneous implants (total dose: 0.2 U/24 h for >30 days, 10 U/kg for 20-g mice). Insulin treatment was continued for 3 wk. Control animals (C; n = 13) received blank implants. Diabetic control was confirmed by biweekly measurements of blood and urinary glucose. In some cases, when urine glucose was present and blood glucose was >288 mg/dl, the animal received a third implant. The insulin treatment was continued until initially lower plasma glucose content in diabetic animals reached control values. Three weeks following implantation, diabetic mice were divided randomly into diabetic-treated (D + I; n = 13) and diabetic-deprived (D ? I; n = 13) groups. Insulin implants were removed from the D ? I group under pentobarbital anesthesia, which led to the return of the diabetic phenotype within 24 h. Animals from the D + I group continued on insulin treatment (Fig. 1). At the age of 18 wk, animals from all groups were analyzed for body composition by an Echo-MRI Body Composition Analyzer (EchoMRI, Houston, TX) and euthanized by decapitation 5 wk after the initial STZ or vehicle dose. Figure 1 depicts the timeline of the experiment and blood glucose profiles for each experimental group. Additional animals were used for estimation of skeletal muscle insulin sensitivity by acute insulin stimulation. The mice were divided into the C (n = 6), D ? I (n = 7), and D + I (n = 7) groups and followed appropriate experimental treatment, except for acute insulin stimulation 10 min prior to euthanization by pentobarbital overdose. Figure 1 of the attached PDF of the article summarizes the study design

Dataset Information

Transcriptomic characterization of the diabetic retinal response to chronic insulin treatment

Publications

Chronic insulin treatment of diabetes does not fully normalize alterations in the retinal transcriptome.

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