Mondo/ChREBP-Mlx-regulated transcriptional network is essential for dietary sugar tolerance in Drosophila - PubMed (original) (raw)
Mondo/ChREBP-Mlx-regulated transcriptional network is essential for dietary sugar tolerance in Drosophila
Essi Havula et al. PLoS Genet. 2013 Apr.
Abstract
Sugars are important nutrients for many animals, but are also proposed to contribute to overnutrition-derived metabolic diseases in humans. Understanding the genetic factors governing dietary sugar tolerance therefore has profound biological and medical significance. Paralogous Mondo transcription factors ChREBP and MondoA, with their common binding partner Mlx, are key sensors of intracellular glucose flux in mammals. Here we report analysis of the in vivo function of Drosophila melanogaster Mlx and its binding partner Mondo (ChREBP) in respect to tolerance to dietary sugars. Larvae lacking mlx or having reduced mondo expression show strikingly reduced survival on a diet with moderate or high levels of sucrose, glucose, and fructose. mlx null mutants display widespread changes in lipid and phospholipid profiles, signs of amino acid catabolism, as well as strongly elevated circulating glucose levels. Systematic loss-of-function analysis of Mlx target genes reveals that circulating glucose levels and dietary sugar tolerance can be genetically uncoupled: Krüppel-like transcription factor Cabut and carbonyl detoxifying enzyme Aldehyde dehydrogenase type III are essential for dietary sugar tolerance, but display no influence on circulating glucose levels. On the other hand, Phosphofructokinase 2, a regulator of the glycolysis pathway, is needed for both dietary sugar tolerance and maintenance of circulating glucose homeostasis. Furthermore, we show evidence that fatty acid synthesis, which is a highly conserved Mondo-Mlx-regulated process, does not promote dietary sugar tolerance. In contrast, survival of larvae with reduced fatty acid synthase expression is sugar-dependent. Our data demonstrate that the transcriptional network regulated by Mondo-Mlx is a critical determinant of the healthful dietary spectrum allowing Drosophila to exploit sugar-rich nutrient sources.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. Drosophila larvae lacking functional Mlx display strong intolerance to dietary sugars.
(A) Schematic presentation of mlx1 mutant locus. P-element is indicated with black arrowhead. (B) Western blot from control and mlx1 mutant larval lysates using anti-Mlx antibodies. (C) Survival of control and mlx1 mutant larvae after 4 days on a 20% sucrose-only diet. (D) Images of control and mlx1 mutants on 20% yeast diet supplemented with 0–15% sucrose after 5, 8, and 13 days after egg laying (dAEL). (E) Pupation kinetics of control and mlx1 mutants on a 20% yeast diet supplemented with 0–15% sucrose. (F) Pupation kinetics and survival of control (tub-GAL4>) and Mlx RNAi (tub-GAL4>Mlx RNAi) larvae on 20% yeast diet with or without 15% sucrose. (G) Rescue of mlx1 mutant pupation on high sugar diet by ubiquitous (tub-GAL4) transgenic expression of mlx. (H) Rescue of adult viability of mlx1 mutants by ubiquitous (tub-GAL4) transgenic expression of mlx. (I) Pupation kinetics of control and mlx1 mutants on a 20% yeast diet supplemented with 10% glucose or fructose. (J) Pupation % of control and mlx1 mutant larvae on pieces of red grape with baker's yeast inoculum. Error bars represent ± SEM (*** p<0.001).
Figure 2. Evidence for functional Mondo-Mlx heterodimer.
(A) Co-purification of Mlx upon pulldown of Strep-tagged Mondo from S2 cells. Mlx was detected on Western blot using anti-Mlx antibodies. Mondo-Strep-V5 was detected using anti-V5 antibodies. Kinesin was used as loading control. (B) Pupation kinetics of control (tub-GAL4>) and Mondo RNAi (tub-GAL4>Mondo RNAi) larvae on 20% yeast diet with or without 15% sucrose. (C) Survival into adulthood of control (tub-GAL4>) and Mondo RNAi (tub-GAL4>Mondo RNAi) animals grown on 20% yeast diet with or without 15% sucrose. Error bars represent ± SEM (** p<0.01).
Figure 3. Changes in lipid and amino acid profiles in mlx1 mutants.
(A) Phospholipid levels in control and mlx1 mutant larvae. PE: phosphatidylethanolamine; LysoPC: lysophosphatidylcholine; PC: phosphatidylcholine. (B) Total triglyceride levels as well as triglycerides divided into two groups based on total fatty acid length. For TG-long chain FA group, total fatty acid carbon number >48. (C) Levels of myristoleic acid and lauric acid were strongly reduced in mlx1 mutant larvae. (D) Ceramide levels in control and mlx1 mutant larvae. (E) Total levels of amino acids in control and mlx1 mutant larvae. (F) Urea levels in control and mlx1 mutant larvae. Error bars represent ± SEM (*p<0.05; ** p<0.01; *** p<0.001). The box contains the middle 50% of the data, the two ends of the boxes represent the upper and the lower quartile of the data. The median is represented by the line in the center of the rectangular box. The vertical lines mark the minimum and maximum data values.
Figure 4. Elevated glucose, trehalose, and glycogen levels in the absence of Mondo-Mlx.
Hemolymph glucose (A) and trehalose (B) levels in control and mlx1 mutant larvae on a 20% yeast diet with or without 5% sucrose. (C) Glycogen levels in control and mlx1 mutant larvae on a 20% yeast-5% sucrose diet. Hemolymph glucose (D) and trehalose (E) levels in control (tub-GAL4>) and Mondo RNAi (tub-GAL4>Mondo RNAi) larvae grown on a 20% yeast-5% sucrose diet. (F) Glycogen levels in control (tub-GAL4>) and Mondo RNAi (tub-GAL4>Mondo RNAi) larvae on 20% yeast-5% sucrose diet. Error bars represent ± SEM (* p<0.05; ** p<0.01; *** p<0.001).
Figure 5. Mlx is functionally important in the fat body.
(A) mRNA expression of mondo and mlx in dissected tissues of 3rd instar larvae measured by quantitative RT-PCR. rp49 was used as a reference gene. (B) Tissue specific rescue of pupation and adult emergence of the mlx1 mutants using neuronal (Elav), muscle (Mef2), or fat body (ppl and r4) –specific GAL4 drivers. (C) Fat body –specific (r4-GAL4) expression of transgenic mlx rescues the elevated circulating glucose in mlx1 mutants. Error bars represent ± SEM (** p<0.01).
Figure 6. Mlx-regulated gene expression program.
(A) Volcano plot indicating genes differentially expressed between control and mlx1 mutant fat bodies. Significant changes (p<0.05 and log2 fold changes>±1) are indicated in orange (upregulated in mlx1) and blue (downregulated in mlx1). (B) Results of a gene-set enrichment analysis (GSEA) of KEGG pathways. Red: upregulated set in mlx1 mutant, blue: downregulated set. The size of nodes illustrates the size of gene sets and the width of edges denotes the overlap between two gene sets. (C) GSEA charts for KEGG categories “Fatty acid metabolism” and “Nitrogen metabolism”. (D) Developmental stage-specific mRNA expression (quantitative-RT-PCR) of selected genes downregulated in mlx1 mutant in the microarray. Expression levels were normalized using Actin 42A as a reference gene. Error bars represent ± SEM (* p<0.05; ** p<0.01; *** p<0.001).
Figure 7. Functional analysis of Mlx-regulated genes.
(A) Pupation kinetics of control (tubts-GAL4>) and Cabut RNAi (tubts-GAL4>Cabut RNAi) larvae on 20% yeast diet with or without 15% sucrose. To avoid early larval lethality, we used temperature sensitive GAL80 with tub-GAL4 system to induce RNAi expression during larval stage. Larvae were maintained in +29°C after collection. Cabut knockdown causes a moderate delay also in the low sugar (20% yeast) diet, but addition of 15% substantially reduces survival and delays pupation further. (B) Pupation kinetics of Aldh-III RNAi (tub-GAL4>Aldh-III RNAi) larvae on 20% yeast diet with or without 15% sucrose. (C) Survival of control (tub-GAL4>) and Aldh-III RNAi (tub-GAL4>Aldh-III RNAi) larvae after 6 days on 20% sucrose-only diet. (D) Survival of control, mlx1, tub-GAL4 and mlx1, tub-GAL4>UAS-Aldh-III larvae after 4 days on a 20% sucrose-only diet. (E) Hemolymph glucose levels in control (tubts-GAL4>) and Cabut RNAi (tubts-GAL4>Cabut RNAi) larvae on 20% yeast-5% sucrose. (F) Hemolymph glucose levels in control (tub-GAL4>) and Aldh-III RNAi (tub-GAL4>Aldh-III RNAi) larvae on 20% yeast-5% sucrose. (G) Total pupation of Fas RNAi (tub-GAL4>Fas RNAi) larvae on 20% yeast diet with or without 15% sucrose. (H) Hemolymph glucose levels in control (tub-GAL4>) and PFK2 RNAi (tub-GAL4>PFK2 RNAi) larvae on 20% yeast-5% sucrose. (I) Pupation kinetics of PFK2 RNAi (tub-GAL4>PFK2 RNAi) larvae on 20% yeast diet with or without 15% sucrose. Error bars represent ± SEM (* p<0.05; ** p<0.01; *** p<0.001).
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This study has been supported by the Academy of Finland, Sigrid Jusélius Foundation, Biocentrum Helsinki, European Research Council, Emil Aaltonen Foundation, Diabetes Research Foundation, Helsinki Graduate Program in Biotechnology and Molecular Biology (EH and MT), Viikki Doctoral Programme in Molecular Biosciences (KH), and University of Helsinki Research Foundation (MT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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