Although the Drosophila melanogaster (fly) model is a popular platform for investigating diet-related phenomena, it can be challenging to measure the volume of agar-based food media flies consume. We addressed this challenge by developing a dye-based method called Consumption-Excretion (Con-Ex). In Con-Ex studies, flies consume solid food labeled with dye, and the volume of food consumed is reflected by the sum of the dye inside of and excreted by flies. Flies consumed-excreted measurable amounts of FD&C Blue No. 1 (Blue 1) and other dyes in Con-Ex studies, but only Blue 1 was readily detectable at concentrations that had no discernable effect on consumption-excretion. In studies with Blue 1, consumption-excretion (i) increased linearly with feeding duration out to 24 h at two different laboratory sites, (ii) was sensitive to starvation, mating status and strain, and (iii) changed in response to alteration of media composition as expected. Additionally, the volume of liquid Blue 1 consumed from capillary tubes was indistinguishable from the volume of Blue 1 excreted by flies, indicating that excreted Blue 1 reflects consumed Blue 1. Our results demonstrate that Con-Ex with Blue 1 as a food tracer is a useful method for assessing ingestion of agar-based food media in adult flies.
Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.
Background Persistent loss of skeletal muscle mass and function as well as altered fat metabolism are frequently observed in severe sepsis survivors. Studies examining sepsis-associated tissue dysfunction from the perspective of the tissue microenvironment are scarce. In this study, we comprehensively assessed transcriptional changes in muscle and fat at single-cell resolution following experimental sepsis induction. Methods Skeletal muscle and visceral white adipose tissue from control mice or mice 1 day or 1 month following faecal slurry-induced sepsis were used. Single cells were mechanically and enzymatically prepared from whole tissue, and viable cells were further isolated by fluorescence activated cell sorting. Droplet-based single-cell RNA-sequencing (scRNA-seq; 10× Genomics) was used to generate single-cell gene expression profiles of thousands of muscle and fat-resident cells. Bioinformatics analyses were performed to identify and compare individual cell populations in both tissues. Results In skeletal muscle, scRNA-seq analysis classified 1438 single cells into myocytes, endothelial cells, fibroblasts, mesenchymal stem cells, macrophages, neutrophils, T-cells, B-cells, and dendritic cells. In adipose tissue, scRNA-seq analysis classified 2281 single cells into adipose stem cells, preadipocytes, endothelial cells, fibroblasts, macrophages, dendritic cells, Bcells, T-cells, NK cells, and gamma delta T-cells. One day post-sepsis, the proportion of most non-immune cell populations was decreased, while immune cell populations, particularly neutrophils and macrophages, were highly enriched. Proportional changes of endothelial cells, neutrophils, and macrophages were validated using faecal slurry and cecal ligation and puncture models. At 1 month post-sepsis, we observed persistent enrichment/depletion of cell populations and further uncovered a cell-type and tissue-specific ability to return to a baseline transcriptomic state. Differential gene expression analyses revealed key genes and pathways altered in post-sepsis muscle and fat and highlighted the engagement of infection/inflammation and tissue damage signalling. Finally, regulator analysis identified gonadotropin-releasing hormone and Bay 11-7082 as targets/compounds that we show can reduce sepsis-associated loss of lean or fat mass. Conclusions These data demonstrate persistent post-sepsis muscle and adipose tissue disruption at the single-cell level and highlight opportunities to combat long-term post-sepsis tissue wasting using bioinformatics-guided therapeutic interventions.
35Cytosolic IDH1 enzyme plays a key, but currently unexplored, role in lipid biosynthesis. Using 36Raman imaging microscopy, we identified heterogeneous lipid profiles in cellular organelles 37 attributed uniquely to IDH1 mutations. Via organelle lipidomics, we found an increase in saturated 38 and monounsaturated fatty acids in the endoplasmic reticulum of IDH1 mut cells compared with 39 IDH WT glioma. We showed that these fatty acids incorporate into phospholipids and induce 40 organelle dysfunctions, with prominent dilation of Golgi apparatus, which can be restored by 41 transient knockdown of stearyl-CoA desaturase or inhibition of D-2-hydroxyglutarate (D-2HG) 42 formation. We validated these findings using tissue from patients with glioma. Oleic acid addition 43 led to increased sensitivity to apoptosis of IDH1 mut cells compared with IDH WT . Addition of D-2HG 44 to U251 WT cells lead in increased ER and Golgi apparatus dilation. Collectively, these studies 45 provide clinically relevant insights into the functional link between IDH1 mut -induced lipid alterations 46 and organelle dysfunction, with therapeutic implications. 48 Significance 52Gliomas are devastating tumors, with the most aggressive form-glioblastoma multiforme-53 correlated with a mean patient survival of 14.5 months. No curative treatment exists to date. Low-54 grade glioma (LGG) with the isocitrate dehydrogenase 1 (IDH1) mutation, R132H, provides a 55 survival benefit to patients. Understanding the unique metabolic profile of IDH1 mut could provide 56 clues regarding its association with longer survival and information about therapeutic targets. 57Herein, we identified lipid imbalances in organelles, generated by IDH mut in cells and patient 58 tissue, that were responsible for Golgi dilation and that correlated with increased survival. Addition 59 3 of oleic acid, which tilted the balance towards elevated levels of monounsaturated fatty acids 60 produced IDH1 mut -specific cellular apoptosis. 65 et al., 2019, Lopez et al., 2010, Victor et al., 2019). IDH1 mutations are an early event, (Lass et 66 al., 2012) are associated with a less aggressive phenotype (Parsons et al., 2008) potentially due 67 to their slow growth and need for nutrients to form D-2-hydroxyglutarate (D-2HG), and are used 68 as prognostic and diagnostic markers of glioma . In fact, the World Health Organization (WHO) 69 released a novel classification of glioma in 2016 to include IDH1 mutations as molecular markers 70 that dictate the classification (Louis et al., 2016). Much effort has been directed toward inhibiting 71 D-2HG formation(Yen et al., 2010, Han and Batchelor, 2017); however, the links between IDH1 72 mutations, tumor metabolism, and clinical manifestation are not well understood.73 Cytosolic NADP-dependent IDH1 plays an important role in lipid biosynthesis via its the 74 production of citrate and NADPH (Koh et al., 2004). The loss of the wildtype (WT) allele in gliomas 75 with the arginine 132 to histidine mutation leads to impaired citrate formation; moreov...
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