The Drosophila HNF4 nuclear receptor promotes glucose-stimulated insulin secretion and mitochondrial function in adults
- University of Utah School of Medicine, United States
Figures
Figure 1 with 2 supplements
dHNF4 mutants are sugar intolerant and display hallmarks of diabetes.
(A) Percent survival of genetically-matched controls and dHNF4 mutants at each stage of development when raised on standard media. Adult viability represents survival past the first day of …
Figure 1—figure supplement 1
Dietary sugar, but not protein, correlates with reduced dHNF4 mutant survival.
Newly hatched first instar larvae were placed in vials (~60 larvae per vial) containing either the 3% sugar diet, the 3% sugar diet + 240 kcal/L extra sugar (2:1 glucose to sucrose, 9% final …
Figure 1—figure supplement 2
Profiling of major metabolites in dHNF4 mutant adults fed different levels of dietary sugar.
Controls and dHNF4 mutants were raised to adulthood on the 3% sugar diet under density-controlled conditions. Five days after eclosion, mature adult males were transferred to the 3%, 9%, or 15% …
Figure 2 with 1 supplement
dHNF4 mutants display defects in glycolysis and mitochondrial metabolism.
GC/MS metabolomic profiling of controls and dHNF4 mutants raised to adulthood on the 3% sugar diet, transferred to the indicated diet for 3 days, and subjected to analysis. Data were obtained from …
Figure 2—figure supplement 1
dHNF4 mutants show broad defects in carbohydrate homeostasis.
GC/MS metabolomic profiling reveals that a range of sugars and sugar alcohols are increased in dHNF4 mutants compared to genetically matched controls, indicating that dHNF4 is required for proper …
Figure 3 with 3 supplements
dHNF4 regulates nuclear and mitochondrial gene expression.
(A) Validation of RNA-seq data by northern blot using total RNA extracted from control and dHNF4 mutant adults. Affected transcripts include those involved in glucose homeostasis (Hex-C, pdgy), the …
Figure 3—figure supplement 1
dHNF4 mutants display changes in TCA cycle intermediates that correlate with changes in gene expression.
GC/MS metabolomic profiling of adult flies fed a sugar only diet (10% sucrose + 1% agar) reveals defective TCA cycle metabolism in dHNF4 mutants. Intermediates of the TCA cycle are presented along …
Figure 3—figure supplement 2
dHNF4 mutants display mitochondrial defects.
(A) mtDNA abundance relative to nuclear DNA quantified by qPCR in control and dHNF4 mutant adult males on the 15% sugar diet. (B) Adult fat body tissue from control or dHNF4 mutants immunostained …
Figure 3—figure supplement 3
Predicted functions of dHNF4 target genes.
Forty-seven genes identified as high confidence targets for direct regulation by dHNF4 are depicted. These genes fit the criteria of showing proximal dHNF4 binding along with reduced transcript …
Figure 4 with 3 supplements
dHNF4 acts through multiple tissues and pathways to control glucose homeostasis.
(A) Circulating glucose levels in adult males expressing tissue-specific RNAi against mCherry (TRiP 35785, grey bars) or dHNF4 (TRiP 29375, dark red bars) in the fat body (r4-GAL4), IPCs (dilp2-GAL4)…
Figure 4—figure supplement 1
dHNF4 is required in the insulin-producing cells and fat body to maintain glucose homeostasis.
(A) Tissue-specific RNAi directed against dHNF4 effectively reduces steady-state levels of dHNF4 protein. Immunostaining was used to detect dHNF4 in organs dissected from third instar larvae that …
Figure 4—figure supplement 2
Fat body-specific disruption of the electron transport chain causes sugar intolerance.
Newly hatched first instar larvae were placed in vials (~60 per vial) containing the 3% or 15% sugar diet and scored for puparium formation when raised at either 25˚C (A) or 18˚C (B). Disruption of …
Figure 4—figure supplement 3
Additional RNAi lines confirming the importance of Hex-C in the fat body for glycemic control.
Tissue-specific RNAi directed against Hex-C in the fat body, but not midgut, results in elevated levels of free glucose. RNAi lines against Hex-C were obtained from the VDRC RNAi collection (UAS-Hex-…
Figure 5 with 1 supplement
dHNF4 is required for glucose-stimulated DILP2 secretion by the insulin-producing cells.
(A) Whole-mount staining for DILP2 peptide in brains dissected from adult control and dHNF4 mutants that were either fasted overnight or re-fed glucose for two hours. (B) Quantification of relative …
Figure 5—figure supplement 1
dHNF4 RNAi in the IPCs causes reduced levels of circulating DILP2-HF.
This data reproduces that shown in Figure 5D using a distinct dHNF4 UAS-RNAi construct (VDRC collection). Levels of circulating HA-FLAG-tagged DILP2 (DILP2HF) were assayed in ad libitum fed animals …
Figure 6
dHNF4 supports a developmental transition toward GSIS and OXPHOS in adult Drosophila.
(A–B) Whole-mount immunostaining of larval (A) or adult (B) brains to detect dHNF4 protein (magenta) or GFP, which marks the IPCs (dilp2>GFP, green). (C) Western blot analysis to detect …
Additional files
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Supplementary file 1
List of genes identified by RNA-seq that display differential abundance between dHNF4 mutant adult males and matched controls, meeting an FDR cutoff of 20 (1%) and Log2 ratio of ± 0.6 (>1.5 fold change).
Transcripts that show reduced abundance in dHNF4 mutants are marked in beige, while those with increased abundance are colored blue.
- https://doi.org/10.7554/eLife.11183.019
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Supplementary file 2
List of the gene ontology categories (determined using GOstat) represented in the top 500 down-regulated and 500 up-regulated genes in dHNF4 mutant adults.
The top 10-16 GO categories for each gene set are listed in order of significance along with the number of genes affected in that category, the total number of genes in that category (in parentheses), and the statistical significance of the match.
- https://doi.org/10.7554/eLife.11183.020
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Supplementary file 3
List of genes with transcription start sites (TSS) within 10 kb of dHNF4 enrichment peaks determined by ChIP-seq analysis, meeting an FDR 20 cutoff (1%).
The chromosomal region and coordinates are indicated for each enrichment peak with neighboring genes listed below each region. Genes are listed from proximal to distal, where the distance reported represents the number of base pairs from the TSS to the middle of the enrichment peak. Gene symbols and corresponding FlyBase gene ID numbers are reported, along with chromosomal location, gene start and stop sites, strand, and TSS position. Neighboring genes also identified by RNA-seq as being either up- or down-regulated in dHNF4 mutants are highlighted in blue and beige, respectively.
- https://doi.org/10.7554/eLife.11183.021
