KLF10 integrates circadian timing and sugar signaling to coordinate hepatic metabolism

  1. Anthony A Ruberto
  2. Aline Gréchez-Cassiau
  3. Sophie Guérin
  4. Luc Martin
  5. Johana S Revel
  6. Mohamed Mehiri
  7. Malayannan Subramaniam
  8. Franck Delaunay
  9. Michèle Teboul  Is a corresponding author
  1. Université Côte d’Azur, CNRS, Inserm, iBV, France
  2. Université Côte d’Azur, CNRS, Institut de Chimie de Nice, France
  3. Department of Biochemistry and Molecular Biology, Mayo Clinic, United States
8 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Genetic disruption of Klf10 in mouse hepatocytes.

(A) Schematic illustrating the strategy used for generating HepKO mice. (B) Relative gene expression of Klf10 mRNA across various tissue in WT and HepKO mice as measured at ZT9 (left) and in WT and …

Figure 1—figure supplement 1
Genetic disruption of Klf10 in mouse hepatocytes.

(A) Body weights (left) and daily food intake (right) of WT and HepKO mice of WT and HepKO mice (mean ± SEM, n = 44 for body weight and n = 6–8 for food intake). (B) Representative actograms of …

Figure 2 with 1 supplement
Deletion of hepatocyte KLF10 alters the circadian transcriptome in the liver.

(A) Schematic illustrating the workflow used to assess the circadian transcriptomes of livers in WT and HepKO mice. (B) Number of rhythmic transcripts detected in the livers from WT and HepKO mice. …

Figure 2—source data 1

MetaCycle analysis of the hepatic circadian transcriptome of WT and HepKO mice fed ad libitum and entrained in a 12:12 light/dark (LD) cycle.

(.xlsx 9.65 MB).

https://cdn.elifesciences.org/articles/65574/elife-65574-fig2-data1-v2.xlsx
Figure 2—source data 2

Phase Set Enrichment Analysis (PSEA) of the hepatic transcriptome of WT and HepKO mice fed ad libitum and entrained in a 12:12 light/dark (LD) cycle.

https://cdn.elifesciences.org/articles/65574/elife-65574-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Deletion of hepatocyte KLF10 alters the circadian transcriptome in the liver.

(A) Gene expression profiles of clock genes in the livers of WT and HepKO mice (n = 3 pools of liver per time point). (B) Gene expression profiles of genes representative of the heatmaps in Figure 2F

Glucose and fructose are potent inducers of Klf10 expression in hepatocytes.

Expression of the Klf10 mRNA in primary WT mouse hepatocytes challenged with low (5 mM) or high (25 mM) glucose (Glu) in the absence or presence of 5 mM fructose (Fru) (mean ± SEM, n = 6) …

Figure 4 with 1 supplement
Loss of hepatocyte KLF10 exacerbates the adverse effects associated with increased sugar consumption.

(A) Schematic illustrating the design of the sugar-sweetened water (SSW) challenge experiment. (B) Gene expression of Klf10 at ZT9 and ZT15 in livers of WT and HepKO mice given a chow or chow + SSW …

Figure 4—figure supplement 1
Loss of hepatocyte KLF10 exacerbates the adverse effects associated with increased sugar consumption.

(A) Daily caloric intake in WT and HepKO mice under the chow and chow + sugar-sweetened water (SSW) dietary conditions (mean ± SEM, n = 8–14). (B) Blood triglyceride content at ZT15 in WT and HepKO …

Figure 5 with 1 supplement
Altered metabolic gene expression in HepKO mice challenged with high sugar.

(A) Expression profiles of Slc2a4 and Pklr. (B) Expression profile of Pck1. (C) Expression profiles of the Fasn and Elovl6. (A–C) Expression was determined at ZT9 and ZT15 in the liver of WT and …

Figure 5—figure supplement 1
Altered metabolic gene expression in HepKO mice challenged with high sugar.

(A) Expression of Slc2a2 and Slc2a5. (B) Expression of Gck, Khk, and G6pc. (C) Expression of Acaca, Acly, Me1, and Thrsp. (A–C) Expression determined at ZT15 in WT and HepKO mice given a chow or …

Figure 6 with 1 supplement
KLF10 governs the transcriptional response to hexose sugars in hepatocytes.

(A) Schematic illustrating the design of the in vitro sugar challenge experiment in WT and HepKO hepatocytes treated with low glucose (LG) or high glucose and fructose (HGF). (B) Representative …

Figure 6—source data 1

Differentially expressed genes in WT and HepKO hepatocytes challenged with high sugar compared to unchallenged cells.

https://cdn.elifesciences.org/articles/65574/elife-65574-fig6-data1-v2.xlsx
Figure 6—source data 2

Kyoto Encyclopedia of Genes and Genomes-enriched pathway in WT and HepKO hepatocytes challenged with high sugar compared to unchallenged cells.

https://cdn.elifesciences.org/articles/65574/elife-65574-fig6-data2-v2.xlsx
Figure 6—source data 3

Values and statistical test results for Figure 6E and Figure 6—figure supplement 1.

https://cdn.elifesciences.org/articles/65574/elife-65574-fig6-data3-v2.xlsx
Figure 6—figure supplement 1
KLF10 governs the transcriptional response to hexose sugars in hepatocytes.

Expression of Elovl6 in WT and HepKO primary hepatocytes treated with low glucose (LG) or high glucose and fructose (HGF) (mean ± SEM, n = 3). Statistics: nonparametric Kruskal–Wallis test. **p<0.01;…

Figure 7 with 1 supplement
KLF10 regulates a large metabolic network in the liver.

(A) Schematic illustrating the workflow used to identify KLF10 bound loci in mouse liver at ZT9. (B) Jaspar logo representing the KLF10 response element DNA sequence and distribution of KLF10 …

Figure 7—source data 1

Genes bound by KLF10 in their –10/+1 kb region or differentially expressed in WT vs. HepKO hepatocytes challenged with high sugar.

https://cdn.elifesciences.org/articles/65574/elife-65574-fig7-data1-v2.xlsx
Figure 7—source data 2

Cytoscape file used to generate, visualize, and analyze the KLF10 regulated metabolic network.

https://cdn.elifesciences.org/articles/65574/elife-65574-fig7-data2-v2.cys
Figure 7—source data 3

Values and statistical test results for Figure 7E and F.

https://cdn.elifesciences.org/articles/65574/elife-65574-fig7-data3-v2.xlsx
Figure 7—figure supplement 1
KLF10 regulates a large metabolic network in liver.

(Top) Immunoblot showing the oscillation of KLF10 protein abundance in the liver of mice entrained in a light/dark (LD) 12:12 cycle. (Bottom) Quantification and cosinor analysis of two independent …

Author response image 1

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus, C57BL/6)WTWeng et al., 2017Klf10flox/flox controls; used males for experiments
Genetic reagent (Mus musculus, C57BL/6)Alb-CreERT2Schuler et al., 2004
Genetic reagent (Mus musculus, C57BL/6)hepKOThis studyUsed males for experiments
Cell line (Mus musculus)WTThis studyPrimary heaptocytes from males; used 150,000–250,000 cells per plate as indicated
Cell line (Mus musculus)hepKOThis studyPrimary hepatocytes from males; used 150,000–250,000 cells per plate as indicated
Biological sample (Mus musculus)Liver, heart, kidney, spleen, lung, white adipose tissueThis study
Biological sample (Mus musculus)BloodThis study
AntibodyAnti-KLF10 (mouse monoclonal)CDI LaboratoriesCat. #m14-355WB (1:800)
AntibodyAnti-EGF1a (mouse monoclonal)Upstate signaling solutionsCat. #05-235WB (1:1000)
AntibodyAnti-IgG (goat polyclonal)SigmaCat. #A4416WB (1:40,000)
Commercial assay or kitGlucose Hexokinase Assay KitSigmaCat. #GAHK20-1KT
Commercial assay or kitPowerUp SYBR green Master MixApplied BiosystemsCat. #A25779
Commercial assay or kitQIAquick PCR Purification KitQiagenCat. #2810
Commercial assay or kitTriglycerides colorimetric assay kitCaymanCat. #10010303
Commercial assay or kitNucleoSpin RNAMacherey-NagelCat. #740955250
Commercial assay or kitRNeasy mini kitQIAGENCat. #74104
Commercial assay or kitNEBNext Ultra RNA Library Prep Kit for IlluminaNew England BiolabsCat. #E7530S
Commercial assay or kitQubitTM dsDNA HS Assay KitInvitrogenCat. #Q32854
Commercial assay or kitGlucose Uptake-Glo AssayPromegaCat. #J1341
Commercial assay or kitMouse Insulin ElisaMercodiaCat. #10-1247-01
Commercial assay or kitGlucose (GOD-PAP)RandoxCat. #8318
Chemical compound, drugDMEM, low glucose, GlutaMAX Supplement, pyruvateGibcoCat. #21885025
Chemical compound, drugDMEM, high glucose, GlutaMAX Supplement, pyruvateGibcoCat. #10569010
Chemical compound, drugDMEM, glucose-freeGibcoCat. #11966025
Chemical compound, drugWilliam’s E mediumGibcoCat. #22551022
Chemical compound, drugDexamethasoneSigmaCat. #D0700000Used 10 nM final concentration
Chemical compound, drugInsulin-selenium-transferrin mixGibcoCat. #41400045Used 1× final concentration
Chemical compound, drugInsulin (human, recombinant zinc)Thermo Fisher ScientificCat. #12585014Used 860 nM final concentration
Chemical compound, drugPercollGE HealthcareCat. #17089101
Chemical compound, drugProtein G DynabeadsThermo Fisher ScientificCat. #10003D
Chemical compound, drugComplete protease inhibitor cocktailRocheCat. #11836153001
Chemical compound, drugCollagenase CLS3WorthingtonCat. #WOLS04180Used 500 U/mL final concentration
Chemical compound, drugCollagenase CLS4WorthingtonCat. #WOLS04186Used 500 U/mL final concentration
Chemical compound, drugAmyloglucosidaseSigmaCat. #11202332001Used 2 mg/mL final concentration
Chemical compound, drugSuperScript II Reverse TranscriptaseInvitrogenCat. #18064014Used 200 U per reaction
Chemical compound, drugMicrococcal nucleaseThermo Fisher ScientificCat. #88216Used 80 U per reaction
Chemical compound, drugProtein G DynabeadsThermo Fisher ScientificCat. #10003D
Software, algorithmRBioconductorhttps://www.bioconductor.org/
Software, algorithmTrimmomaticBolger et al., 2014http://www.usadellab.org/cms/
Software, algorithmFastQCBabraham Institutehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/
Software, algorithmFASTQ GroomerGalaxy projecthttps://sourceforge.net/projects/fastqgroomer/
Software, algorithmBowtie2Langmead and Salzberg, 2012https://github.com/BenLangmead/bowtie2RRID: SCR_016368
Software, algorithmBEDtoolsQuinlan and Hall, 2010https://github.com/arq5x/bedtools2RRID: SCR_006646
Software, algorithmRsubread (featureCounts)Liao et al., 2014https://git.bioconductor.org/packages/RsubreadRRID: SCR_009803
Software, algorithmMetaCycleWu et al., 2016https://github.com/gangwug/MetaCycleApp
Software, algorithmPhase Set Enrichment AnalysisZhang et al., 2016https://github.com/ranafi/PSEA
Software, algorithmMACS2Feng et al., 2012https://pypi.org/project/MACS2/
Software, algorithmGREATMcLean et al., 2010http://great.stanford.edu/public/html/splash.php
Software, algorithmFIMOBailey et al., 2015http://meme-suite.org/tools/fimo
Software, algorithmFastHeinzBeisser et al., 2010https://www.bioconductor.org/packages/release/bioc/html/BioNet.html
Software, algorithmclusterMaker2Morris et al., 2011http://www.rbvi.ucsf.edu/cytoscape/clusterMaker2/
Software, algorithmBingoMaere et al., 2005https://github.com/cytoscape/BiNGO
Software, algorithmCytoscapeShannon et al., 2003https://cytoscape.org/
OtherQubit fluorometerThermo Fisher ScientificCat. #Q33238
OtherFreeStyle InsuLinx glucometerAbbottN/A
OtherLaboratory rodent chow dietSafe dietsCat. #R03-25
OtherApistar syrup (organic)ICKOCat. #HC406

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