The role of endothelial insulin signaling in the regulation of vascular tone and insulin resistance

Abstract

Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: insulin receptor (IR); endothelin-1 (ET-1); vascular endothelial cell insulin receptor knockout (VENIRKO); glucose-tolerance test (GTT); insulintolerance tests (ITT); systolic blood pressure (SBP); mean blood pressure (MBP); diastolic blood pressure (DBP); heart rate (HR); glucose infusion rate (GIR); lipoprotein lipase (LPL). these animals and explore the role of the insulin receptor in retinal neovascularization. MethodsGeneration of mice and genotyping. VENIRKO mice were generated using the Cre-loxP system. Mice carrying an IR gene in which exon 4 is flanked by lox sites (20) were bred with a transgenic mice expressing Cre recombinase under control of the Tie2 promoter-enhancer. Previous studies have shown that Tie2 expression is limited to vascular endothelial cells and the endocardial cushion (21). As a result of the complex breeding, all the mice in these experiments have a mixed genetic background, including contributions from 129Sv, C57Bl/6, SJL, FVB, and DBA strains. To minimize the difference of genetic background, we used littermates from same breeding pairs as controls. Genotyping of the mice for the Cre transgene and the floxed IR alleles was performed by genomic PCR, using specific primers that allow identification of the presence or absence of the Tie2-Cre transgene and heterozygosity or homozygosity of the IR gene for the floxed or wild-type allele (22). Animals were housed on a 12-h light/dark cycle and were fed a standard rodent chow. All protocols for animal use and euthanasia were reviewed and approved by the Animal Care Committee of the Joslin Diabetes Center and were in accordance with NIH guidelines. All experiments were carried out in male mice, except as otherwise noted.Endothelial culture and evaluation of the Cre-mediated recombination of IR in vascular endothelial cells. Two approaches were used to derive microvascular endothelium for primary culture. Cerebrovascular microvessels were isolated by filtration of brain homogenates and digestion with collagenase (23). The digested vessels were filtered sequentially through nylon meshes of three different pore sizes 210, 88, and 55 µm, respectively. The microvessels retained on 88-and 55-µm filters were plated on 30-mm fibronectin-coated dishes and incubated in DMEM medium containing 10% FCS and 2% bovine brain EGF for 7-10 days. Nonendothelial cells were scraped off using a cotton bar under microscopic visualization. Cultures that were considered more than 95% pure based on morphological examination were used to prepare genomic DNA. Cre-mediated recombination of IR gene was analyzed by PCR using primers surrounding exon 4, as described previously (22).To obtain enough endothelial cells to prepare RNA for real-time quantitative RT-PCR, primary cultures of endothelial cells were established using cells immunoselected from lungs (24). Briefly, a cell suspension was prepared from lungs by digestion with collagenase (1 mg/ml) for...

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Molecular and physiologic actions of insulin related to production of nitric oxide in vascular endothelium

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