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  • Diacylglycerol kinase was one of the hits identified from


    Diacylglycerol kinase was one of the hits identified from the RNAi screen. Diacylglycerol kinases are conserved across a Lenalidomide of species with more complex, multicellular organisms possessing several DGKs with differing protein domains, expression patterns and functions [61], [62], [63]. Some of these functions include metabolically-relevant ones including central control of energy homeostasis and insulin resistance [64], [65], [66]. For example, type I DGKs have been associated with insulin secretion from pancreatic β-cells in vitro and Lenalidomide SNPs near DGKB and DGKG have been associated with obesity related-measures by genome-wide association studies. However, little is known about the in vivo role of type I DGKs in metabolism. In Drosophila, there is a single type I DGK whose function is largely uncharacterized. Using multiple lines of evidence, we found that Dgk plays an essential role in regulating energy homeostasis by acting within the IPCs in the CNS to regulate secretion of dILP2 and dILP5. First, the TAG, glucose, and glycogen phenotypes seen with knockdown or overexpression of Dgk using fru-Gal4 can be recapitulated with dIlp2-Gal4. These findings are consistent with the association of SNPs near Dgk homologues with BMI, weight and fasting blood glucose levels. Furthermore, manipulations of Dgk levels affect dILP2 and dILP5 levels in the hemolymph at the level of secretion since dIlp2 and dIlp5 transcript levels are unchanged. However, it is possible that Dgk could affect dILP protein stability or trafficking into secretory vesicles and remains to be measured. This Dgk-mediated disturbance in dILP secretion alters insulin signalling activity, which is likely responsible for the changes in lipid and sugar energy stores. While a similar function has been suggested for its mammalian homologues in cultured pancreatic β-cells, our results constitute the first in vivo confirmation of this function as well as its physiological consequences. The mechanism through which Dgk affects dILP secretion still needs to be determined. The large majority of functions attributed to DGKs are dependent on their kinase function and its regulation of cellular DAG and PA levels [61], [62], [63], both of which are important signalling molecules. There are a few studies that implicate DAG and PA in the regulation of insulin secretion: DAG activates PKCs [67], [68], [69] and Munc13, a synaptic protein that regulates vesicle release [70], while PA increases insulin granule trafficking and exocytosis [71], [72]. It is therefore possible, that Dgk functions to regulate the cellular levels of these two signalling molecules in the insulin-producing cells which in turn, modulate dILP secretion. However, a possible kinase-independent function of Dgk in energy homeostasis is supported by our results from overexpression of wild-type and kinase-dead Dgk. With the exception of low TAG levels, overexpression of wild-type Dgk did not exhibit any defects in the other measured phenotypes. Instead, overexpression of kinase-dead DgkG509D gave many phenotypes that might be expected with wild-type Dgk i.e. opposing phenotypes compared to Dgk RNAi. These differences are not due to reduced expression of wild-type Dgk protein. There have been several instances of other DGKs where the enzymatic activity is not required for its function in specific contexts [73], [74], [75], [76], [77]. Thus, the exact mechanism through which Dgk regulates dILP secretion in Drosophila remains to be determined. Interestingly, despite high circulating dILP2 and dILP5 levels in fru > Dgk RNAi flies, overall insulin signalling pathway activity was attenuated suggesting that these flies are insulin resistant. These results are consistent with previous studies that showed that flies, like mammals, develop insulin resistance in conjunction with increased TAG and sugar levels [78], [79], [80].
    Acknowledgments This work was supported by a grant from the Canadian Institutes of Health (FRN97871) and a Tier I Canada Research Chair in Molecular and Developmental Neurobiology to G.L.B. I.T. is the recipient of a University of Toronto Open Fellowship and Hospital for Sick Children Research Training Competition award.