406. rAAV-Mediated Expression of Wnt10b cDNA Prevents the Development of Obesity in DIO Rats

Wnt10b is a new recently described member of secreted type glyco-proteins family Wnt. It is classified as the canonical Wnt to activate the β-catenin-TCF pathway and regulates diverse developmental processes.The purpose of this study was to investigate long-term metabolic effects of the Wnt10b in diet-induced obese (DIO) rat model. Sustained expression was achieved by delivering mouse Wnt10b cDNAs via a viral vector. To this end, we have generated recombinant adeno-associated virus (rAAV) vector of serotype 1 encoding Wnt10b cDNAs by using baculovirus expression system.Vector was first tested on immortalized fibroblast cell line (3T3- L1) that differentiate to adipocytes recapitulating metabolic and endocrine functions of adipocytes in vivo. Cells were infected by rAAV1-Wnt10b or rAAV1-GFP (negative control) and than induced to differentiate by using the respective hormone cocktail. We have shown that rAAV-mediated Wnt10b completely blocked differentiation of 3T3-L1 fibroblasts to adipocyte in vitro.The long-term effect of rAAV-mediated Wnt10b on adipogenesis in vivo was tested upon peripheral injection in diet-induced obese (DIO) female rats fed high fat (60%) diet. We have shown that a single im injection of 1x1012 physical particles of rAAV1-Wnt10b resulted in sustained (up to 120 days) statistically significant (p = 0.0000007) reduction in body weight (BW) accumulation with attendant reduction in hyperinsulinemia (p = 0.0006) and triglycerides plasma level (p = 0.0000009). This insulin-enhancing effect was substantiated by IP Glucose Tolerance Test (IP GTT), and was concomitant with the decreases levels of intramyocellular lipids (IMCLs) in the skeletal muscles shown by H-MRS. Distribution of white fat and reduction of visceral white fat pad was measured by MRI. Hormonal profiles were monitored using Enzyme-Linked Immunosorbent Assays (ELISA). The molecular mechanism of weight-reducing effect of transgene Wnt10b expression was found to be a Wnt10b downstream signal transduction inhibiting the expression of major adipogenic transcription factors, C/EBP-alpha and PPAR-gamma, as well as the adipocyte gene FABP-4. The study demonstrated a strong therapeutic effect of rAAV-mediated ectopic expression of Wnt10b on BW and insulin sensitivity.