Overexpression of Calreticulin in endothelial cells changes insulin trascytosis

Carneiro-Lobo T.C., Dalloul R.S.D., Mesaeli N. Department of Biochemistry, Weill Cornell Medical College in Qatar, Doha, Qatar. Introduction: The incidence of diabetes and obesity has been rising world wide in the past few decades. In the Gulf Cooperation Council (GCC) a rapid increase in the prevalence of diabetes has been reposted in the past few years. In Qatar the incidence of diabetes is 16.7% in the Qatari population that is almost 3 times higher than the incidence of diabetes in UK. Type 2 diabetes mellitus is a growing public health problem and a major cause of cardiovascular disease in the United State. Type 2 diabetes is associated with systemic insulin resistance, which promotes hyperglycemia, and it has been proposed that these metabolic abnormalities account for increased cardiovascular risk. Endothelial dysfunction contributes to the pathogenesis and clinical expression of atherosclerosis and has been linked to type 2 diabetes mellitus and insulin resistance. Transport of insulin across the microvasculature is necessary to reach its target organs and is rate limiting in insulin action. The two possible mechanism of this movement is either through leaky tight junction in vascular endothelial cell layer or via transcytosis through these endothelial cells. Therefore, we hypothesized that overexpression of calreticulin would reduce insulin transport to the target tissue due to onset of endothelial dysfunction. Methods and Results: To test our hypothesis, we used genetic approaches to overexpress calreticulin (CRT). For in vitro experiments CRT was overexpressed using a Lentiviral-CRT-RFP to infect Human Umbilical Vein Endothelial Cells (HUVEC). For ex vivo studies we used endothelial cels isolated from our endothelial specific CRT overexpressing transgenic mice (ECCRT+). We examined the uptake of fluorescently labeled insulin in the endothelial cells. CRT-HUVEC and WT-HUVEC were incubated with 25 μg of FITC-insulin for 30 mins and 60 mins. We found that CRT-overexpressing cells reduce the transcytosis of insulin through the endothelial cells compared to control. The primary endothelial cells were isolated using columns containing PECAM (CD31) coated beads. Cells were then characterized by detection of CD31 (PECAM) and Von Willebrand factor (vWF) expression by immunofluorescence and western blot. After confirming the endothelial identity of our primary cells (ECCRT+) they were incubated with Alexa-647 insulin for 30 min or 60 min. Confocal microscopy was carried out to examine insulin uptake and transcytosis. Our data illustrate a reduction in insulin trancytosis in ECCRT+ endothelial cells as compared with WT cells. To evaluate how Calreticulin affects insulin trancytosis in vivo in whole animal, ECCRT+ mice and WT-mice were used. Alexaflour-647 insulin (1.2 μg/L) was injected via tail vein of ECCRT+ mouse and WT-mouse. To label the surface of endothelial cells, 20 μg/L FITC-isolectin was injected via tail vein following insulin injection. 30 mins after the injection mice were euthanized and tissue (lung, liver, heart and retina) were isolated, fixed and embedded for cryosections. Tissue sections were then mounted on slides and examined by confocal microscopy. We observed a lower concentration of insulin in cell membrane of endothelial cells of vascular wall in lung, liver, heart and retina of ECCRT+ mouse than WT-mice. Finally, we examined the leakiness of the vascular wall in our ECCRT+ mouse model. In these experiemnts we injected 2.5 mg/ml FITC-Dextran in the tail vein for 30 min. After euthaniasia, mice tissue was imaged under a Ziess flourescnet microscope to evaluate leakage of the flourescet signal in the tissue. Fluorescent images from the lung, liver and heart were collected and illustrated an increase in the leakiness of vascular wall of the small vessels in the ECCRT+ mice as compared to the WT mice. Conclusion: Taken together, our results illustrate that increased calreticulin expression in endothelial cells affect endothelial cell function, increase the vascular permeability and reduce the transcytosis of insulin through the endothelial cell of vasculature. The impaired insulin export from the vascular wall to the target tissue could contribute to the onset of insulin resistance and diabetes in the ECCRT+ mice which we have observed in our other studies on these mice. This is the first report to link calreticulin expression level and insulin transport in intact animal. Acknowledgment: This research was made possible by a grant by QNRF, NPRP07-208-3-046.