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Excess nutrients are stored as triglycerides, mostly as lipid droplets found in adipose tissue. Previous studies have characterized a group of splicing factors called serine/arginine rich (SR) proteins that function to identify intron/exon borders in regulating metabolic homeostasis in the Drosophila fat body. Decreasing the function of one SR protein, 9G8, causes an increase in triglyceride storage; however, the full complement of genes regulated by 9G8 to control metabolism is unknown. To address this question, we performed RNA sequencing on Drosophila fat bodies with 9G8 levels reduced by RNAi. Differential expression and differential exon usage analyses revealed several genes involved in the immune response, xenobiotic biology, protein translation, sleep, and lipid and carbohydrate metabolism whose expression or splicing is altered in 9G8-RNAi fat bodies. One gene that was both downregulated and had altered splicing in 9G8-RNAi fat bodies was Zwischenferment (Zw), the Drosophila homolog of human glucose 6-phosphate dehydrogenase (G6PD). G6PD regulates flux of glucose 6-phosphate (G6P) into the pentose phosphate pathway, which generates NADPH, a coenzyme for lipid synthesis. Interestingly, the other NADPH-producing enzyme genes in Drosophila (phosphogluconate dehydrogenase, isocitrate dehydrogenase and malic enzyme) were also decreased in 9G8-RNAi flies. Together, these findings suggest that 9G8 regulates several classes of genes and may regulate NADPH-producing enzyme genes to maintain metabolic homeostasis.

After a meal, excess nutrients are stored within adipose tissue as triglycerides in structures called lipid droplets. Previous genome-wide RNAi screens have identified that mRNA splicing factor genes are required for normal lipid droplet formation in Drosophila cells. We have previously shown that mRNA splicing factors called serine/arginine-rich (SR) proteins are important for triglyceride storage in the Drosophila fat body. SR proteins shuttle in and out of the nucleus with the help of proteins called Transportins (Tnpo-SR); however, whether this transport is important for SR protein-mediated regulation of lipid storage is unknown. The purpose of this study is to characterize the role of Tnpo-SR proteins in regulating lipid storage in the Drosophila fat body. Decreasing Tnpo-SR in the adult fat body resulted in an increase in triglyceride storage and consistent with this phenotype, Tnpo-SR-RNAi flies also have increased starvation resistance. In addition, the lipid accumulation in Tnpo-SR-RNAi flies is the result of increased triglyceride stored in each fat body cell and not due to increased food consumption. Interestingly, the splicing of CPT1, an enzyme important for the β-oxidation of fatty acids, is altered in Tnpo-SR-RNAi fat bodies. The isoform that produces the less catalytically active form of CPT1 accumulates in fat bodies where Tnpo-SR levels are decreased, suggesting a decrease in lipid breakdown, potentially causing the excess triglyceride storage observed in these flies. Together, these data suggest that the transport of splicing proteins in and out of the nucleus is important for proper triglyceride storage in the Drosophila fat body.

Polyamines are low molecular weight, organic cations that play a critical role in many major cellular processes including cell cycle regulation and apoptosis, cellular division, tissue proliferation, and cellular differentiation; however, the functions of polyamines in regulating the storage of metabolic fuels such as triglycerides and glycogen is poorly understood. To address this question, we focused on the Drosophila homolog of ornithine decarboxylase (Odc1), the first rate-limiting enzyme in the synthesis of polyamines. Mutants in Odc1 are lethal, but heterozygotes were viable to adulthood. Odc1 heterozygotes appeared larger than their genetic background control flies and consistent with this observation, weighed more than the controls. However, the increased weight was not due to increased food consumption as heterozygotes ate less than the controls. Interestingly, Odc1 heterozygous flies had augmented triglyceride storage, and this lipid phenotype was due to increased triglyceride storage per cell and an increase in the number of fat cells produced. Odc1 heterozygous flies also displayed increased expression of the lipid synthesis genes fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), suggesting increased lipid synthesis was the cause of the augmented triglyceride phenotype. These results provide a link between the expression of Odc1 and triglyceride storage suggesting that the polyamine pathway plays a role in regulating lipid metabolism.

Excess triglycerides from the diet are stored in structures called lipid droplets in adipose tissue. Genome-wide RNAi screens have identified mRNA splicing factors as important for lipid droplet formation; however, the full complement of splicing factors that regulate lipid storage is not known. Here, we characterize the role of

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit. We found, in general, that the attitudes students bring with them into the classroom contribute to two outcome measures, namely, learning as assessed by a pre- and postquiz and perceived self-reported benefits. While the GEP CURE produces positive outcomes overall, the students with more positive attitudes toward science, particularly with respect to epistemic beliefs, showed greater gains. The findings indicate the importance of a student's epistemic beliefs to achieving positive learning outcomes.

Polyamines are small organic cations that are important for several biological processes such as cell proliferation, cell cycle progression, and apoptosis. The dysregulation of intracellular polyamines is often associated with diseases such as cancer, diabetes, and developmental disorders. Although polyamine metabolism has been well studied, the effects of key enzymes in the polyamine pathway on lipid metabolism are not well understood. Here, we determined metabolic effects resulting from the absence of spermidine synthase (SpdS) and spermine synthase (Sms) in Drosophila. While SpdS mutants developed normally and accumulated triglycerides, Sms mutants had reduced viability and stored less triglyceride than the controls. Interestingly, when decreasing SpdS and Sms, specifically in the fat body, triglyceride storage increased. While there was no difference in triglycerides stored in heads, thoraxes and abdomen fat bodies, abdomen fat body DNA content increased, and protein/DNA decreased in both SpdS- and Sms-RNAi flies, suggesting that fat body-specific knockdown of SpdS and Sms causes the production of smaller fat body cells and triglycerides to accumulate in non-fat body tissues of the abdomen. Together, these data provide support for the role that polyamines play in the regulation of metabolism and can help enhance our understanding of polyamine function in metabolic diseases.

The availability of excess nutrients in Western diets has led to the overaccumulation of these nutrients as triglycerides, a condition known as obesity. The full complement of genes important for regulating triglyceride storage is not completely understood. Genome-wide RNAi screens in Drosophila cells have identified genes involved in mRNA splicing as important lipid storage regulators. Our lab has shown that a group of splicing factors called heterogeneous nuclear ribonucleoproteins (hnRNPs) regulate lipid metabolism in the fly fat body; however, the identities of all the hnRNPs that function to control triglyceride storage are not known. Here, we used the GAL4/UAS system to induce RNAi to the hnRNP glorund (glo) in the Drosophila fat body to assess whether this hnRNP has any metabolic functions. Decreasing glo levels resulted in less triglycerides being stored throughout the fly. Interestingly, decreasing fat body glo expression resulted in increased triglyceride storage in the fat body, but blunted triglyceride storage in non-fat body tissues, suggesting a defect in lipid transport. Consistent with this hypothesis, the expression of apolipophorin (apolpp), microsomal triglyceride transfer protein (mtp), and apolipoprotein lipid transfer particle (apoltp), apolipoprotein genes important for lipid transport through the fly hemolymph, was decreased in glo-RNAi flies, suggesting that glo regulates the transport of lipids from the fly fat body to surrounding tissues. Together, these results indicate that glorund plays a role in controlling lipid transport and storage and provide additional evidence of the link between gene expression and the regulation of lipid metabolism., Highlights • The metabolic functions of the hnRNP glorund (glo) are unknown. • Fat body glo-RNAi increases fat body triglyceride storage. • Fat body glo-RNAi decreases triglyceride storage in non-fat body tissues. • Decreasing glo in the fly fat body blunts the expression of lipid transport genes.

A hallmark of the research experience is encountering experimental difficulty and working through that challenge to reach success. The ability to overcome scientific challenges is essential to being a successful scientist, but replicating these challenges can be difficult in a teaching setting. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students’ learning experience in this research is continuous and recursive, frequently beginning with frustration but eventually leading to success. In order to explore our “formative frustration” hypothesis, we gathered data about this research experience from faculty via a survey, and from students both via a general survey and a small set of focus groups administered at the end of the GEP CURE. All three datasets contained comments mentioning frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of what we have deemed “formative frustration” is an important aspect for a successful CURE.

Metabolic state is a potent modulator of sleep and circadian behavior, and animals acutely modulate their sleep in accordance with internal energy stores and food availability. Across phyla, hormones secreted from adipose tissue act in the brain to control neural physiology and behavior to modulate sleep and metabolic state. Growing evidence suggests the fat body is a critical regulator of complex behaviors, but little is known about the genes that function within the fat body to regulate sleep. To identify molecular factors functioning in non-neuronal tissues to regulate sleep, we performed an RNAi screen selectively knocking down genes in the fat body. We found that knockdown of Phosphoribosylformylglycinamidine synthase/Pfas (Ade2), a highly conserved gene involved the biosynthesis of purines, sleep regulation and energy stores. Flies heterozygous for multiple Ade2 mutations are also short sleepers and this effect is partially rescued by restoring Ade2 to the Drosophila fat body. Targeted knockdown of Ade2 in the fat body does not alter arousal threshold or the homeostatic response to sleep deprivation, suggesting a specific role in modulating baseline sleep duration. Together, these findings suggest Ade2 functions within the fat body to promote both sleep and energy storage, providing a functional link between these processes.

The storage of excess nutrients as triglycerides is essential for all organisms to survive when food is scarce; however, the mechanisms by which triglycerides are stored are not completely understood. Genome-wide RNAi screens in Drosophila cells have identified genes involved in mRNA splicing that are important in the regulation of triglyceride storage. Our lab has identified a number of splicing factors important for regulating lipid metabolism; however, the full complement of splicing proteins involved in achieving metabolic homeostasis is unknown. Heterogeneous nuclear ribonucleoproteins (hnRNPs), RNA binding proteins that inhibit the splicing of introns by preventing the assembly of splicing complexes, have no established metabolic functions. To assess any metabolic functions of hnRNPs, we used the GAL4/UAS system to induce RNAi to six hnRNP's: hnRNP-K, rumpelstiltskin (rump), smooth (sm), Hrb27C (also referred to as Hrp48), Hrb98DE, and Hrb87F in the Drosophila fat body. Decreasing the levels of hnRNP-K and rump resulted in a decrease in triglyceride storage, whereas decreasing the levels of sm, Hrb27C, and Hrb98DE resulted in an increase in triglyceride storage. The excess triglyceride phenotype in Hrb27C-RNAi flies resulted from both an increase in the number of fat body cells and the amount of fat stored per cell. In addition, both the splicing of the β-oxidation gene, CPT1, and the expression of the lipase brummer (bmm) was altered in flies with decreased Hrb27C, providing insight into the lipid storage phenotype in these flies. Together, these results suggest that the hnRNP family of splicing factors have varying metabolic functions and may act on specific metabolic genes to control their expression and processing.

Regulation of sleep and metabolic homeostasis is critical to an animal's survival and under stringent evolutionary pressure. Animals display remarkable diversity in sleep and metabolic phenotypes; however, an understanding of the ecological forces that select for, and maintain, these phenotypic differences remains poorly understood. The fruit fly, Drosophila melanogaster, is a powerful model for investigating the genetic regulation of sleep and metabolic function, and screening in inbred fly lines has led to the identification of novel genetic regulators of sleep. Nevertheless, little is known about the contributions of naturally occurring genetic differences to sleep, metabolic phenotypes, and their relationship with geographic or environmental gradients. Here, we quantified sleep and metabolic phenotypes in 24 D. melanogaster populations collected from diverse geographic localities. These studies reveal remarkable variation in sleep, starvation resistance, and energy stores. We found that increased sleep duration is associated with proximity to the equator and elevated average annual temperature, suggesting that environmental gradients strongly influence natural variation in sleep. Further, we found variation in metabolic regulation of sleep to be associated with free glucose levels, while starvation resistance associates with glycogen and triglyceride stores. Taken together, these findings reveal robust naturally occurring variation in sleep and metabolic traits in D. melanogaster, providing a model to investigate how evolutionary and ecological history modulate these complex traits.

Excess nutrients are stored as triglycerides mainly in the adipose tissue of an animal and these triglycerides are located in structures called lipid droplets. Previous genome-wide RNAi screens in Drosophila cells identified splicing factors as playing a role in lipid droplet formation. Our lab has recently identified the SR protein, 9G8, as an important factor in fat storage as decreasing its levels results in augmented triglyceride storage in the fat body. Previous in vitro studies have implicated 9G8 in the regulation of splicing of the sex determination gene doublesex (dsx) by binding to transformer (tra) and transformer2 (tra2); however, any function of these sex determination proteins in regulating metabolism is unknown. In this study, we have uncovered a role of tra2 to regulate fat storage in vivo. Inducing tra2dsRNA in the adult fat body resulted in an increase in triglyceride levels but had no effect on glycogen storage. Consistent with the triglyceride phenotype, tra2 knockdown flies lived longer under starvation conditions. In addition, this increase in triglycerides is due to more fat storage per cell and not an increase in the number of fat cells. Interestingly, the splicing of CPT1, an enzyme involved in the breakdown of lipids, was altered in flies with decreased tra2. The less-catalytically active isoform of CPT1 accumulated in tra2dsRNA flies suggesting a decrease in lipid breakdown, which is consistent with the increased triglyceride levels observed in these flies. Together, these results suggest a link between mRNA splicing, sex determination and lipid metabolism and may provide insight into the mechanisms underlying tissue-specific splicing and nutrient storage.

Polyamines are small, naturally occurring polycations that are essential for normal cell growth and development. Maintaining a homeostatic level of both the polyamine pathway enzymes and the levels of the polyamines putrescine, spermidine and spermine is important because the dysregulation of the polyamine pathway has been associated with physical anomalies including cancer, Parkinson's disease, Snyder-Robinson syndrome and aging in mammals. To date, little is understood regarding how the polyamine pathway maintains polyamine homeostasis in the fly, an emerging model for polyamine studies. The aim of these studies was to better characterize how the downregulation or ablation of the polyamine biosynthetic enzymes ornithine decarboxylase, spermidine synthase and spermine synthase influence the expression of the genes coding for other polyamine pathway enzymes and the levels of the polyamines themselves utilizing the model organism, Drosophila melanogaster. We demonstrate the complexities of the polyamine metabolic pathway and show that downregulation of any of these polyamine pathway enzymes can lead to an array of changes in other parts of the pathway as well as alter the cellular levels of polyamines themselves. These data highlight the complex nature of regulation of this pathway and underscore the need to better understand its self-regulation in this and other model systems.

The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (∼5.2 Mb) is the smallest chromosome in Drosophila melanogaster, but it is substantially larger (>18.7 Mb) in D. ananassae. To identify the major contributors to the expansion of the F element and to assess their impact, we improved the genome sequence and annotated the genes in a 1.4-Mb region of the D. ananassae F element, and a 1.7-Mb region from the D element for comparison. We find that transposons (particularly LTR and LINE retrotransposons) are major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F-element genes exhibit distinct characteristics compared to D-element genes (e.g., larger coding spans, larger introns, more coding exons, and lower codon bias), but these differences are exaggerated in D. ananassae. Compared to D. melanogaster, the codon bias observed in D. ananassae F-element genes can primarily be attributed to mutational biases instead of selection. The 5′ ends of F-element genes in both species are enriched in dimethylation of lysine 4 on histone 3 (H3K4me2), while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F-element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves our understanding of how transposons can affect genome size and how genes can function within highly repetitive domains.

In Western societies where food is abundant, these excess nutrients are stored as fats mainly in adipose tissue. Fats are stored in structures known as lipid droplets, and a genome-wide screen performed in Drosophila cells has identified several genes that are important for the formation of these droplets. One group of genes found during this screen included those that regulate mRNA splicing. Previous work from our lab has identified some splicing factors that play a role in regulating fat storage; however, the full complement of splicing proteins that regulate lipid metabolism is still unknown. In this study, the levels of a number of serine-arginine (SR) domain containing splicing factors (RSF1, RBP1, RBP1-like, SF2 and Srp-54) were decreased using RNAi in the adult fat body to assess their role in the control of Drosophila metabolism. Decreasing SF2 and RBP1 showed increased triglycerides, while inducing RNAi towards RSF1, RBP1-Like and Srp-54 had no effect on triglycerides. Interestingly, the increased triglyceride phenotype in the SF2-RNAi flies was due to an increase in the amount of fat stored per cell while the RBP1-RNAi flies have more fat cells. In addition, the splicing of the β-oxidation enzyme, CPT1, was altered in the SF2-RNAi flies potentially promoting the increased triglycerides in these animals. Together, this study identifies novel splicing factors responsible for the regulation of lipid storage in the Drosophila fat body and contributes to our understanding of the mechanisms, which influence the regulation of fat storage in adipose-like cells.

Many metabolic consequences of the excess lipid storage observed in obesity have been documented in humans and mammals, but the effects of an obese phenotype on reproductive behavior are less understood. Drosophila melanogaster (Meigan, 1830) is well suited to study the ramifications of excess energy storage as they are commonly used to explore the genes important for energy utilization. However, little is known about the reproductive behavior of mutants in these metabolic genes. Therefore, we tested, for the first time, courtship of the naturally occurring adipose (adp60) mutation which accumulates excess triglycerides and glycogen. Adipose mutants showed decreased courtship behaviors as well as copulation rate. The decrease in copulation rate could in part be explained by the decrease in courtship behaviors such as wing scissoring and wing vibration. Taken together, the data presented here suggest that the adp60 mutants may negatively affect male fly reproductive behavior and provide a system to further our understanding of the genetic and metabolic control of reproductive behaviors and reproductive potential.

Dysregulation of sleep or feeding has enormous health consequences. In humans, acute sleep loss is associated with increased appetite and insulin insensitivity, while chronically sleep-deprived individuals are more likely to develop obesity, metabolic syndrome, type II diabetes, and cardiovascular disease. Conversely, metabolic state potently modulates sleep and circadian behavior; yet, the molecular basis for sleep-metabolism interactions remains poorly understood. Here, we describe the identification of translin (trsn), a highly conserved RNA/DNA binding protein, as essential for starvation-induced sleep suppression. Strikingly, trsn does not appear to regulate energy stores, free glucose levels, or feeding behavior suggesting the sleep phenotype of trsn mutant flies is not a consequence of general metabolic dysfunction or blunted response to starvation. While broadly expressed in all neurons, trsn is transcriptionally upregulated in the heads of flies in response to starvation. Spatially restricted rescue or targeted knockdown localizes trsn function to neurons that produce the tachykinin family neuropeptide Leucokinin. Manipulation of neural activity in Leucokinin neurons revealed these neurons to be required for starvation-induced sleep suppression. Taken together, these findings establish trsn as an essential integrator of sleep and metabolic state, with implications for understanding the neural mechanism underlying sleep disruption in response to environmental perturbation.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Regulation of sleep and metabolic homeostasis are critical to an animal’s survival and under stringent evolutionary pressure. Animals display remarkable diversity in sleep and metabolic phenotypes; however, an understanding of the ecological forces that select for, and maintain, these phenotypic differences remain poorly understood. The fruit fly,Drosophila melanogaster, is a powerful model for investigating the genetic regulation of sleep and metabolic function, and screening in inbred fly lines has led to the identification of novel genetic regulators of sleep. Nevertheless, little is known about the contributions of naturally occurring genetic differences to sleep, metabolic phenotypes, and their relationship with geographic or environmental gradients. Here, we quantified sleep and metabolic phenotypes in 24D. melanogasterpopulations collected from unique geographic localities. These studies reveal remarkable diversity in sleep, starvation resistance, and energy stores. We found that increased sleep duration is strongly associated with proximity to the equator and elevated average annual temperature, suggesting that environmental gradients strongly influence natural variation in sleep. Further, we found variation in metabolic regulation of sleep to be associated with free glucose levels, while starvation resistance associates with glycogen and triglyceride stores. Taken together, these findings reveal robust naturally occurring variation in sleep and metabolic traits inD. melanogasterand suggest that distance from the equator and median temperature is a significant evolutionary factor in sleep regulation and architecture.

Dysregulation of sleep is associated with metabolic diseases, and metabolic rate is acutely regulated by sleep-wake behavior. In humans and rodent models, sleep loss is associated with obesity, reduced metabolic rate, and negative energy balance, yet little is known about the neural mechanisms governing interactions between sleep and metabolism. We have developed a system to simultaneously measure sleep and metabolic rate in individual Drosophila, allowing for interrogation of neural systems governing interactions between sleep and metabolic rate. Like mammals, metabolic rate in flies is reduced during sleep and increased during sleep deprivation suggesting sleep-dependent regulation of metabolic rate is conserved across phyla. The reduction of metabolic rate during sleep is not simply a consequence of inactivity because metabolic rate is reduced ∼30 minutes following the onset of sleep, raising the possibility that CO2 production provides a metric to distinguish different sleep states in the fruit fly. To examine the relationship between sleep and metabolism, we determined basal and sleep-dependent changes in metabolic rate is reduced in starved flies, suggesting that starvation inhibits normal sleep-associated effects on metabolic rate. Further, translin mutant flies that fail to suppress sleep during starvation demonstrate a lower basal metabolic rate, but this rate was further reduced in response to starvation, revealing that regulation of starvation-induced changes in metabolic rate and sleep duration are genetically distinct. Therefore, this system provides the unique ability to simultaneously measure sleep and oxidative metabolism, providing novel insight into the physiological changes associated with sleep and wakefulness in the fruit fly.Significance statementMetabolic disorders are associated with sleep disturbances, yet our understanding of the mechanisms underlying interactions between sleep and metabolism remain limited. Here, we describe a novel system to simultaneously record sleep and metabolic rate in single Drosophila. Our findings reveal that uninterrupted sleep bouts of 30 minutes or greater are associated with a reduction in metabolic rate providing a physiological readout of sleep. Use of this system, combined with existing genetic tools in Drosophila, will facilitate identification of novel sleep genes and neurons, with implications for understanding the relationship between sleep loss and metabolic disease.

Dysregulation of sleep and metabolism has enormous health consequences. Sleep loss is linked to increased appetite and insulin insensitivity, and epidemiological studies link chronic sleep deprivation to obesity-related disorders including type II diabetes and cardiovascular disease. Interactions between sleep and metabolism involve the integration of signaling from brain regions regulating sleep, feeding, and metabolic function. Investigating the relationship between these processes provides a model to address more general questions of how the brain prioritizes homeostatically regulated behaviors. The availability of powerful genetic tools in the fruit fly, Drosophila melanogaster, allows for precise manipulation of neural function in freely behaving animals. There is a strong conservation of genes and neural circuit principles regulating sleep and metabolic function, and genetic screens in fruit flies have been effective in identifying novel regulators of these processes. Here, we review recent findings in the fruit fly that further our understanding of how the brain modulates sleep in accordance with metabolic state.

While course-based research in genomics can generate both knowledge gains and a greater appreciation for how science is done, a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. Nonetheless, this is a very cost-effective way to reach larger numbers of students., There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge. We have developed a bioinformatics project that provides a course-based research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. An alumni survey revealed that time spent on a research project is also a significant factor in the value former students assign to the experience one or more years later. We conclude: 1) implementation of a bioinformatics project within the biology curriculum provides a mechanism for successfully engaging large numbers of students in undergraduate research; 2) benefits to students are achievable at a wide variety of academic institutions; and 3) successful implementation of course-based research experiences requires significant investment of instructional time for students to gain full benefit.

The storage of lipids is an evolutionarily conserved process that is important for the survival of organisms during shifts in nutrient availability. Triglycerides are stored in lipid droplets, but the mechanisms of how lipids are stored in these structures are poorly understood. Previous in vitro RNAi screens have implicated several components of the spliceosome in controlling lipid droplet formation and storage, but the in vivo relevance of these phenotypes is unclear. In this study, we identify specific members of the splicing machinery that are necessary for normal triglyceride storage in the Drosophila fat body. Decreasing the expression of the splicing factors U1-70K, U2AF38, U2AF50 in the fat body resulted in decreased triglyceride levels. Interestingly, while decreasing the SR protein 9G8 in the larval fat body yielded a similar triglyceride phenotype, its knockdown in the adult fat body resulted in a substantial increase in lipid stores. This increase in fat storage is due in part to altered splicing of the gene for the β-oxidation enzyme CPT1, producing an isoform with less enzymatic activity. Together, these data indicate a role for mRNA splicing in regulating lipid storage in Drosophila and provide a link between the regulation of gene expression and lipid homeostasis.

Sleep length and metabolic dysfunction are correlated, but the causal relationship between these processes is unclear. Octopamine promotes wakefulness in the fly by acting through the insulin-producing cells (IPCs) in the fly brain. To determine if insulin signaling mediates the effects of octopamine on sleep:wake behavior, we assayed flies in which insulin signaling activity was genetically altered. We found that increasing insulin signaling does not promote wake, nor does insulin appear to mediate the wake-promoting effects of octopamine. Octopamine also affects metabolism in invertebrate species, including, as we show here, Drosophila melanogaster. Triglycerides are decreased in mutants with compromised octopamine signaling and elevated in flies with increased activity of octopaminergic neurons. Interestingly, this effect is mediated at least partially by insulin, suggesting that effects of octopamine on metabolism are independent of its effects on sleep. We further investigated the relative contribution of metabolic and sleep phenotypes to the starvation response of flies with altered octopamine signaling. Hyperactivity (indicative of foraging) induced by starvation was elevated in octopamine receptor mutants, despite their high propensity for sleep, indicating that their metabolic state dictates their behavioral response under these conditions. Moreover, flies with increased octopamine signaling do not suppress sleep in response to starvation, even though they are normally hyper-aroused, most likely because of their high triglyceride levels. Together, these data suggest that observed correlations between sleep and metabolic phenotypes can result from shared molecular pathways rather than causality, and environmental conditions can lead to the dominance of one phenotype over the other.

SummaryCircadian rhythms are regulated by a synchronized system of central and peripheral clocks. Here, we show that a clock in the Drosophila fat body drives rhythmic expression of genes involved in metabolism, detoxification, the immune response, and steroid hormone regulation. Some of these genes cycle even when the fat body clock is disrupted, indicating that they are regulated by exogenous factors. Food is an important stimulus, as limiting food availability to a 6 hr interval each day drives rhythmic expression of genes in the fat body. Restricting food to a time of day when consumption is typically low desynchronizes internal rhythms because it alters the phase of rhythmic gene expression in the fat body without affecting the brain clock. Flies maintained on this paradigm produce fewer eggs than those restricted to food at the normal time. These data suggest that desynchrony of endogenous rhythms, caused by aberrant feeding patterns, affects reproductive fitness.

Animals recognize the availability of nutrients and regulate the intake and storage of these nutrients accordingly. However, the molecular mechanisms underlying nutrient sensing and subsequent changes in behavior and metabolism are not fully understood. Mlx interactor (Mio), the Drosophila homolog of carbohydrate response element binding protein (ChREBP), functions as a transcription factor in the fat body of the fly to control triglyceride storage as well as feeding, suggesting that Mio may act in a nutrient-sensing pathway to coordinate food consumption and metabolism. Here, we show that Mio functions in neurons in Drosophila to regulate feeding and nutrient storage. Pan-neuronal disruption of Mio function leads to increased triglyceride and glycogen storage, and this phenotype is not due to increased food consumption. Interestingly, targeted disruption of Mio specifically in the insulin-producing cells (IPCs) has little effect on nutrient storage, but increases food consumption suggesting that Mio acts in these neurons to control feeding behavior. Since Mio is a transcription factor, one possible way Mio may act in the IPCs to control feeding is through regulating the expression of Drosophila insulin-like peptides (dilps) or drosulfakinin (dsk), neuropeptides produced in the IPCs. Consistent with this hypothesis, IPC-specific knockdown of Mio leads to an increase in dilp3 expression, while not affecting dilp2, 5 or dsk levels. Together, this study indicates a new function for Mio in the Drosophila brain and specifically in the IPCs, controlling neuropeptide gene expression, feeding and metabolism in accordance with nutrient availability.

Initial studies have suggested that Pop1/Bves protein is exclusively expressed in the smooth muscle walls of the coronary vessels, implying its possible importance in coronary diseases. However, the mRNA and activity of this gene are detected in both skeletal and cardiac muscles, not coronary smooth muscle, and Pop1/Bves knockout mice have defects in skeletal muscle regeneration. Here we used specific monoclonal antibodies (MAbs) raised against chicken Pop1/Bves and demonstrated the presence of this protein in cardio-myocytes through development and its apparent absence in coronary vessels. Immunostaining of cardiomyocytes cultured in vitro confirmed the membrane localization of this protein in cells that participate in cell adhesion, with significant intracellular staining seen in isolated cells. In skeletal muscle, Pop1 protein becomes detectable at embryonic day (E) 7, coincident with the differentiation of morphologically distinct muscle masses from the limb muscle blastema, but the protein is not found at high levels in the cell membrane of myotubes until E11, coincident with the formation of secondary myotubes from satellite cells. These data support the hypothesis that Pop1/Bves is a cell adhesion molecule present in skeletal and cardiac muscle.

There have been numerous calls to engage students in science as science is done. A survey of 90-plus faculty members explores barriers and incentives when developing a research-based genomics course. The results indicate that a central core supporting a national experiment can help overcome local obstacles., In their 2012 report, the President's Council of Advisors on Science and Technology advocated “replacing standard science laboratory courses with discovery-based research courses”—a challenging proposition that presents practical and pedagogical difficulties. In this paper, we describe our collective experiences working with the Genomics Education Partnership, a nationwide faculty consortium that aims to provide undergraduates with a research experience in genomics through a scheduled course (a classroom-based undergraduate research experience, or CURE). We examine the common barriers encountered in implementing a CURE, program elements of most value to faculty, ways in which a shared core support system can help, and the incentives for and rewards of establishing a CURE on our diverse campuses. While some of the barriers and rewards are specific to a research project utilizing a genomics approach, other lessons learned should be broadly applicable. We find that a central system that supports a shared investigation can mitigate some shortfalls in campus infrastructure (such as time for new curriculum development, availability of IT services) and provides collegial support for change. Our findings should be useful for designing similar supportive programs to facilitate change in the way we teach science for undergraduates.

During nutrient excess, triglycerides are synthesized and stored to provide energy during times of famine. The presence of high glucose leads to the activation of carbohydrate response element binding protein (ChREBP), a transcription factor that induces the expression of a number of glycolytic and lipogenic enzymes. ChREBP is expressed in major metabolic tissues and while we have a basic understanding of ChREBP function in liver, in vivo genetic systems to study the function of ChREBP in other tissues are lacking. In this study, we characterized the role of the Drosophila homolog of ChREBP, Mlx interactor (Mio), in controlling fat accumulation in larvae and adult flies. In Mio mutants, high sugar-induced lipogenic enzyme mRNA expression is blunted and lowering Mio levels specifically in the fat body using RNA interference leads to a lean phenotype. A lean phenotype is also observed when the gene bigmax, the fly homolog of ChREBP’s binding partner Mlx, is decreased in the larval fat body. Interestingly, depleting Mio in the fat body results in decreased feeding providing a potential cause of the lowered triglycerides observed in these animals. However, Mio does not seem to function as a general regulator of hunger-induced behaviors as decreasing fat body Mio levels has no effect on sleep under fed or starved conditions. Together, these data implicate a role for Mio in controlling fat accumulation in Drosophila and suggests that it may act as a nutrient sensor in the fat body to coordinate feeding behavior with nutrient availability.

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.

Innate immunity is the primary and most ancient defense against infection. Although critical to survival, coordinating protection against a foreign organism is energetically costly, creating the need to reallocate substrates from nonessential functions, such as growth and nutrient storage. However, the mechanism by which infection or inflammation leads to a reduction in energy utilization by these dispensable processes is not well understood. Here, we demonstrate that activation of the Toll signaling pathway selectively in the fat body, the major immune and lipid storage organ of the fruit fly, Drosophila melanogaster , leads to both induction of immunity and reallocation of resources. Toll signaling in the fat body suppresses insulin signaling both within these cells and non-autonomously throughout the organism, leading to a decrease in both nutrient stores and growth. These data suggest that communication between these two regulatory systems evolved as a means to divert energy in times of need from organismal growth to the acute requirement of combating infection.

A phylogenetically conserved response to nutritional abundance is an increase in insulin signaling, which initiates a set of biological responses dependent on the species. Consequences of augmented insulin signaling include developmental progression, cell and organ growth, and the storage of carbohydrates and lipids. Here, we address the evolutionary origins of insulin's positive effects on anabolic lipid metabolism by selectively modulating insulin signaling in the fat body of the fruit fly, Drosophila melanogaster. Analogous to the actions of insulin in higher vertebrates, those in Drosophila include expansion of the insect fat cell mass both by increasing the adipocyte number and by promoting lipid accumulation. The ability of insulin to accomplish the former depends on its capacity to bring about phosphorylation and inhibition of the transcription factor Drosophila FOXO (dFOXO) and the serine/threonine protein kinase shaggy, the fly ortholog of glycogen synthase kinase 3 (GSK3). Increasing the amount of triglyceride per cell also depends on the phosphorylation of shaggy but is independent of dFOXO. Thus, the findings of this study provide evidence that the control of fat mass by insulin is a conserved process and place dFOXO and shaggy/GSK3 downstream of the insulin receptor in controlling adipocyte cell number and triglyceride storage, respectively.

All cells require energy to perform their specialized functions. Muscle is particularly sensitive to the availability of nutrients due to the high-energy requirement for muscle contraction. Therefore the ability of muscle cells to obtain, store and utilize energy is essential for the function of these cells. Mio, the Drosophila homolog of carbohydrate response element binding protein (ChREBP), has recently been identified as a nutrient responsive transcription factor important for triglyceride storage in the fly fat body. However, the function of Mio in muscle is unknown. In this study, we characterized the role of Mio in controlling muscle function and metabolism. Decreasing Mio levels using RNAi specifically in muscle results in increased thorax glycogen storage. Adult Mio-RNAi flies also have a flight defect due to altered myofibril shape and size in the indirect flight muscles as shown by electron microscopy. Myofibril size is also decreased in flies just before emerging from their pupal cases, suggesting a role for Mio in myofibril development. Together, these data indicate a novel role for Mio in controlling muscle structure and metabolism and may provide a molecular link between nutrient availability and muscle function.

Pop1 (BVES) is a member of the Popeye gene family which was named for its high-level expression in the heart and other muscle lineages. However, these proteins have no sequence similarity to any other gene family and their function is unclear. Here we report the production of recombinant chicken Pop1/BVES protein and the generation of two Pop1/BVES specific monoclonal antibodies (MAbs) 3F11-D9-E8 and 1B3-G11-A8. These antibodies detect recombinant Pop1/BVES in ELISAs and endogenous chicken Pop1/BVES in chicken heart extracts by Western blotting. Further, both 3F11-D9-E8 and 1B3-G11-A8 detect Pop1/BVES specifically in the cardiomyocytes of 8-day-old embryonic chicken hearts by immunofluorescence. These MAbs will be useful in immunolocalization and immunoblotting experiments of different tissue types to determine the location and levels of Pop1/BVES expression throughout development, as well as further analysis of the biochemistry of this protein.