Probing the mechanisms of signal transduction via adiponectin and its receptors

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DESCRIPTION (provided by applicant): Obesity correlates with the development of type 2 diabetes, however, the molecular mechanisms behind this correlation are inadequately characterized. The adipocyte-derived anti-diabetic hormone, adiponectin, has emerged as a promising candidate for such a molecular link. Low circulating adiponectin and low adiponectin receptor (AdipoR1 and AdipoR2) expression correlate with insulin resistance. Therefore, a detailed characterization of how adiponectin receptors transmit signals is critical for a better understanding of how obesity results insulin resistance and for the future development of therapeutics for type 2 diabetes that target the physiology of adiponectin. Current theories of the mechanism by which adiponectin receptors transmit signals inside the cell suggest that they function as a novel type of G-protein-coupled receptor. This grant proposal challenges that assignment and hypothesizes that these receptors function, instead, as adiponectin-activated ceramidase enzymes. Furthermore, it is proposed that these receptors, via the generation of a sphingosine second messenger, activate PDK1 and downstream AGC kinase pathways known to be involved in glucose sensing (protein kinase A, protein kinase B or AMP-dependent protein kinase). To confirm this hypothesis, three specific aims are proposed using the yeast, Saccharomyces cerevisiae, as a model system. Working in yeast is beneficial for several reasons. First, the pathways of sphingolipid-dependent signal transduction and glucose signaling in humans are highly conserved in yeast. Second, AdipoR1 has been shown to function when expressed heterologously in yeast. And third, the physiological effects of AdipoR1 expression in yeast can be readily investigated due to the ease of experimentation in this organism. In Specific Aim 1, the sphingolipid- and adiponectin receptor-dependent activation of the yeast homologues of PDK1 and downstream AGC kinases will be confirmed by measuring their phosphorylation state and by measuring the effect of kinase inactivation on a promoter-reporter construct that is known to respond to AdipoR1 overexpression. In Specific Aim 2, the effect of adiponectin receptor expression on in vivo sphingolipid metabolism will be confirmed. And lastly, in Specific Aim 3, the ceramidase activity of the adiponectin receptors will be confirmed by in vitro biochemical assay. This hypothesis, if proven correct, will redirect research into the mechansims of adiponectin signaling.