The Central Clock Neurons Regulate Lipid Storage in Drosophila

Citation: DiAngelo JR, Erion R, Crocker A, Sehgal A ( The Central Clock Neurons Regulate Lipid Storage in Drosophila Justin R. DiAngelo 0 Renske Erion 0 Amanda Crocker 0 Amita Sehgal 0 Stuart E. Dryer, University of Houston, United States of America 0 1 Department of Neuroscience, The University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2 Howard Hughes Medical Institute, Hofstra University , Hempstead , New York, United States of America, 3 Department of Biology, Hofstra University , Hempstead, New York , United States of America A proper balance of lipid breakdown and synthesis is essential for achieving energy homeostasis as alterations in either of these processes can lead to pathological states such as obesity. The regulation of lipid metabolism is quite complex with multiple signals integrated to control overall triglyceride levels in metabolic tissues. Based upon studies demonstrating effects of the circadian clock on metabolism, we sought to determine if the central clock cells in the Drosophila brain contribute to lipid levels in the fat body, the main nutrient storage organ of the fly. Here, we show that altering the function of the Drosophila central clock neurons leads to an increase in fat body triglycerides. We also show that although triglyceride levels are not affected by age, they are increased by expression of the amyloid-beta protein in central clock neurons. The effect on lipid storage seems to be independent of circadian clock output as changes in triglycerides are not always observed in genetic manipulations that result in altered locomotor rhythms. These data demonstrate that the activity of the central clock neurons is necessary for proper lipid storage. - Funding: This work was supported by National Institutes of Health grants T32-AG000255, which provided support for JRD, F31-MH080490 to AC. AS is an investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Throughout evolution, the ability of humans to convert glucose to triglyceride for long-term storage has provided a competitive advantage during times of famine. However, in our current Western society where food is abundantly available, this thrifty phenotype has resulted in excess fat accumulation leading to 65% of adults in the United States being overweight and 30% being obese [1]. Clearly, a proper balance of the synthesis and breakdown of lipids is essential for reaching metabolic homeostasis, but the mechanisms responsible for controlling these processes are still not fully understood. The regulation of lipid metabolism is a very complex process, utilizing a number of signals and pathways leading to lipid synthesis, breakdown or both [2]. Recent research has focused on understanding the regulation of lipid metabolism in liver and adipose tissue by the brain (reviewed in [3,4]). In mammals, the arcuate nucleus (ARC) of the hypothalamus serves as a main regulator of energy homeostasis by integrating signals from many circulating hormones. The ARC also receives neural inputs from other regions of the hypothalamus, one of these being the suprachiasmatic nucleus (SCN), the site of the central circadian clock [5]. The circadian system is, in fact, known to be a major regulator of metabolic activity, with profound metabolic phenotypes reported in clock mutant animals [6,7]. However, analysis of underlying mechanisms has focused on autonomous effects of clocks located in metabolic tissues such as the control of gene expression by such clocks as well as interactions between clock proteins and metabolic factors in these tissues [8,9,10]. Despite the connection between the ARC and the SCN, little is known about the contribution of the central clock to metabolic processes. The fruit fly, Drosophila melanogaster, is a well-established model of circadian rhythms and has recently become a powerful model to study the regulation of metabolism [11]. In Drosophila, as in mammals, the central clock is found in specific neurons of the brain, but clocks also exist in other body tissues [12,13,14]. However, effects of these different clocks on metabolic activity are poorly understood. We showed recently that the Drosophila fat body (equivalent of mammalian liver and adipose tissue) contains a circadian clock, which regulates the storage of glycogen and triglycerides [15]. Clocks in neurons also affect glycogen storage, but the specific neurons responsible were not identified and the control of triglyceride levels by neuronal clocks was not assessed [15]. Here, we sought to explore a role of the central clock neurons in the accumulation of lipids. We report that knocking down the function of the circadian gene, Clock (Clk) in central clock cells leads to increased triglycerides in the flys fat body. We observe a similar phenotype when we trigger premature degeneration in these neurons. However, triglyceride levels are normal in arrhythmic flies that express the heat-sensitive ion channel dTRPA1 in the PDF neurons and in Pdf01 mutants, suggesting that these neurons control fat storage independently of the circadian rest:activity output. In addition, over-expression of the clock gene, timeless (tim), in these neurons does not affect triglycerides although it reduces behavioral rhythmicity. Together, these findings indicate a non-circadian Materials and Methods Fly genetics Flies were grown on standard cornmeal molasses medium at room temperature. Prior to each experiment, 03 day old females were entrained for 23 days in a 12 h:12 h light:dark cycle at 25uC. For dTRPA1 experiments, flies were reared at 1821uC and 03 day old flies were shifted to 27uC in 12 h:12 h light:dark conditions for seven days before being assayed. Fly strains used in this study include: iso31 (Bloomington #5905), ClkJrk [16], cyc01 [17], tim01 [18], per0 [19], Pdf-Gal4 [20], UAS-ClkD [14], UASClkRNAi (VDRC #42834), UAS-Ab42ArcM [21], UAS-tim [22], UAS-dTRPA1 [23] and Pdf01 [20]. Triglyceride and protein measurements Fat bodies were dissected from abdomens of 47d old mated females as described previously [24]. Fat bodies were homogenized in lysis buffer containing 140 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.1% Triton X and 1X protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and triglyceride and protein measurements were made using the triglyceride LiquiColor kit (Stanbio Laboratory, Boerne, TX) and bicinchoninic acid protein assay kit (Thermo Scientific, Waltham, MA), respectively, according to manufacturers instructions. Single balancer chromosomes were used to identify Gal4 and transgene only control flies for these experiments; since the single balancer chromosomes had no difference in triglycerides compared to wildtype chromosomes (data not shown), both wildtype and balancer chrom (...truncated)