Jill L. Fink

A Drosophila p38 orthologue is required for environmental stress responses

The p38 mitogen‐activated protein kinase (MAPK) cascade is an evolutionarily conserved signalling mechanism involved in processes as diverse as apoptosis, cell fate determination, immune function and stress response. Aberrant p38 signalling has been implicated in many human diseases, including heart disease, cancer, arthritis and neurodegenerative diseases. To further understand the role of p38 in these processes, we generated a Drosophila strain that is null for the D‐p38a gene. Mutants are homozygous viable and show no observable developmental defects. However, flies lacking D‐p38a are susceptible to some environmental stresses, including heat shock, oxidative stress and starvation. These phenotypes only partially overlap those caused by mutations in D‐MEKK1 and dTAK1, suggesting that the D‐p38a gene is required to mediate some, but not all, of the functions ascribed to p38 signalling.

Dmp53 protects the Drosophila retina during a developmentally regulated DNA damage response

Ultraviolet (UV) light is absorbed by cellular proteins and DNA, promoting skin damage, aging and cancer. In this paper, we explore the UV response by cells of the Drosophila retina. We demonstrate that the retina enters a period of heightened UV sensitivity in the young developing pupa, a stage closely associated with its period of normal developmental programmed cell death. Injury to irradiated cells included morphology changes and apoptotic cell death; these defects could be completely accounted for by DNA damage. Cell death, but not morphological changes, was blocked by the caspase inhibitor P35. Utilizing genetic and microarray data, we provide evidence for the central role of Hid expression and for Diap1 protein stability in controlling the UV response. In contrast, we found that Reaper had no effect on UV sensitivity. Surprisingly, Dmp53 is required to protect cells from UV‐mediated cell death, an effect attributed to its role in DNA repair. These in vivo results demonstrate that the cellular effects of DNA damage depend on the developmental status of the tissue.

Role of Fat Body Lipogenesis in Protection against the Effects of Caloric Overload in Drosophila

Background: A high sugar diet leads to obesity and insulin resistance in Drosophila. Results: The metabolic fate of dietary glucose is reprogrammed in high sugar-fed and lean animals. Conclusion: Obesity is protective against the deleterious effects of a high sugar diet. Significance: An emerging perspective that obesity is protective against sequelae of human metabolic disease is conserved in the fly. The Drosophila fat body is a liver- and adipose-like tissue that stores fat and serves as a detoxifying and immune responsive organ. We have previously shown that a high sugar diet leads to elevated hemolymph glucose and systemic insulin resistance in developing larvae and adults. Here, we used stable isotope tracer feeding to demonstrate that rearing larvae on high sugar diets impaired the synthesis of esterified fatty acids from dietary glucose. Fat body lipid profiling revealed changes in both carbon chain length and degree of unsaturation of fatty acid substituents, particularly in stored triglycerides. We tested the role of the fat body in larval tolerance of caloric excess. Our experiments demonstrated that lipogenesis was necessary for animals to tolerate high sugar feeding as tissue-specific loss of orthologs of carbohydrate response element-binding protein or stearoyl-CoA desaturase 1 resulted in lethality on high sugar diets. By contrast, increasing the fat content of the fat body by knockdown of king-tubby was associated with reduced hyperglycemia and improved growth and tolerance of high sugar diets. Our work supports a critical role for the fat body and the Drosophila carbohydrate response element-binding protein ortholog in metabolic homeostasis in Drosophila.

A Drosophila functional evaluation of candidates from human genome-wide association studies of type 2 diabetes and related metabolic traits identifies tissue-specific roles for dHHEX

BackgroundGenome-wide association studies (GWAS) identify regions of the genome that are associated with particular traits, but do not typically identify specific causative genetic elements. For example, while a large number of single nucleotide polymorphisms associated with type 2 diabetes (T2D) and related traits have been identified by human GWAS, only a few genes have functional evidence to support or to rule out a role in cellular metabolism or dietary interactions. Here, we use a recently developed Drosophila model in which high-sucrose feeding induces phenotypes similar to T2D to assess orthologs of human GWAS-identified candidate genes for risk of T2D and related traits.ResultsDisrupting orthologs of certain T2D candidate genes (HHEX, THADA, PPARG, KCNJ11) led to sucrose-dependent toxicity. Tissue-specific knockdown of the HHEX ortholog dHHEX (CG7056) directed metabolic defects and enhanced lethality; for example, fat-body-specific loss of dHHEX led to increased hemolymph glucose and reduced insulin sensitivity.ConclusionCandidate genes identified in human genetic studies of metabolic traits can be prioritized and functionally characterized using a simple Drosophila approach. To our knowledge, this is the first large-scale effort to study the functional interaction between GWAS-identified candidate genes and an environmental risk factor such as diet in a model organism system.

CoA protects against the deleterious effects of caloric overload in Drosophila

We developed a Drosophila model of T2D in which high sugar (HS) feeding leads to insulin resistance. In this model, adipose TG storage is protective against fatty acid toxicity and diabetes. Initial biochemical and gene expression studies suggested that deficiency in CoA might underlie reduced TG synthesis in animals during chronic HS feeding. Focusing on the Drosophila fat body (FB), which is specialized for TG storage and lipolysis, we undertook a series of experiments to test the hypothesis that CoA could protect against the deleterious effects of caloric overload. Quantitative metabolomics revealed a reduction in substrate availability for CoA synthesis in the face of an HS diet. Further reducing CoA synthetic capacity by expressing FB-specific RNAi targeting pantothenate kinase (PK orfumble) or phosphopantothenoylcysteine synthase (PPCS) exacerbated HS-diet-induced accumulation of FFAs. Dietary supplementation with pantothenic acid (vitamin B5, a precursor of CoA) was able to ameliorate HS-diet-induced FFA accumulation and hyperglycemia while increasing TG synthesis. Taken together, our data support a model where free CoA is required to support fatty acid esterification and to protect against the toxicity of HS diets.

A high throughput, functional screen of human Body Mass Index GWAS loci using tissue-specific RNAi Drosophila melanogaster crosses

Human GWAS of obesity have been successful in identifying loci associated with adiposity, but for the most part, these are non-coding SNPs whose function, or even whose gene of action, is unknown. To help identify the genes on which these human BMI loci may be operating, we conducted a high throughput screen in Drosophila melanogaster. Starting with 78 BMI loci from two recently published GWAS meta-analyses, we identified fly orthologs of all nearby genes (± 250KB). We crossed RNAi knockdown lines of each gene with flies containing tissue-specific drivers to knock down (KD) the expression of the genes only in the brain and the fat body. We then raised the flies on a control diet and compared the amount of fat/triglyceride in the tissue-specific KD group compared to the driver-only control flies. 16 of the 78 BMI GWAS loci could not be screened with this approach, as no gene in the 500-kb region had a fly ortholog. Of the remaining 62 GWAS loci testable in the fly, we found a significant fat phenotype in the KD flies for at least one gene for 26 loci (42%) even after correcting for multiple comparisons. By contrast, the rate of significant fat phenotypes in RNAi KD found in a recent genome-wide Drosophila screen (Pospisilik et al. (2010) is ~5%. More interestingly, for 10 of the 26 positive regions, we found that the nearest gene was not the one that showed a significant phenotype in the fly. Specifically, our screen suggests that for the 10 human BMI SNPs rs11057405, rs205262, rs9925964, rs9914578, rs2287019, rs11688816, rs13107325, rs7164727, rs17724992, and rs299412, the functional genes may NOT be the nearest ones (CLIP1, C6orf106, KAT8, SMG6, QPCTL, EHBP1, SLC39A8, ADPGK /ADPGK-AS1, PGPEP1, KCTD15, respectively), but instead, the specific nearby cis genes are the functional target (namely: ZCCHC8, VPS33A, RSRC2; SPDEF, NUDT3; PAGR1; SETD1, VKORC1; SGSM2, SRR; VASP, SIX5; OTX1; BANK1; ARIH1; ELL; CHST8, respectively). The study also suggests further functional experiments to elucidate mechanism of action for genes evolutionarily conserved for fat storage.

Seven-Up Is a Novel Regulator of Insulin Signaling

Musselman et al. address the overarching question: “What’s so bad about a high-calorie diet?” Using computational biology to analyze mRNA expression profiles, the authors built a Drosophila fat body gene regulatory network that predicted... Insulin resistance is associated with obesity, cardiovascular disease, non-alcoholic fatty liver disease, and type 2 diabetes. These complications are exacerbated by a high-calorie diet, which we used to model type 2 diabetes in Drosophila melanogaster. Our studies focused on the fat body, an adipose- and liver-like tissue that stores fat and maintains circulating glucose. A gene regulatory network was constructed to predict potential regulators of insulin signaling in this tissue. Genomic characterization of fat bodies suggested a central role for the transcription factor Seven-up (Svp). Here, we describe a new role for Svp as a positive regulator of insulin signaling. Tissue-specific loss-of-function showed that Svp is required in the fat body to promote glucose clearance, lipid turnover, and insulin signaling. Svp appears to promote insulin signaling, at least in part, by inhibiting ecdysone signaling. Svp also impairs the immune response possibly via inhibition of antimicrobial peptide expression in the fat body. Taken together, these studies show that gene regulatory networks can help identify positive regulators of insulin signaling and metabolic homeostasis using the Drosophila fat body.