1. Biochemistry and Chemical Biology
  2. Developmental Biology

Hormone-sensitive lipase couples intergenerational sterol metabolism to reproductive success

  1. Christoph Heier  Is a corresponding author
  2. Oskar Knittelfelder
  3. Harald F Hofbauer
  4. Wolfgang Mende
  5. Ingrid Pörnbacher
  6. Laura Schiller
  7. Gabriele Schoiswohl
  8. Hao Xie
  9. Sebastian Grönke
  10. Andrej Shevchenko
  11. Ronald P Kühnlein
  1. Institute of Molecular Biosciences, University of Graz, Austria
  2. BioTechMed-Graz, Austria
  3. Max Planck Institute of Molecular Cell Biology and Genetics, Germany
  4. Field of Excellence BioHealth - University of Graz, Austria
  5. Max Planck Institute for Biophysical Chemistry, Germany
https://doi.org/10.7554/eLife.63252
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Cite this article
  1. Christoph Heier
  2. Oskar Knittelfelder
  3. Harald F Hofbauer
  4. Wolfgang Mende
  5. Ingrid Pörnbacher
  6. Laura Schiller
  7. Gabriele Schoiswohl
  8. Hao Xie
  9. Sebastian Grönke
  10. Andrej Shevchenko
  11. Ronald P Kühnlein
(2021)
Hormone-sensitive lipase couples intergenerational sterol metabolism to reproductive success
eLife 10:e63252.
https://doi.org/10.7554/eLife.63252
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7 figures, 3 tables and 2 additional files

Figures

Figure 1
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Enzyme activities and subcellular localization of Drosophila Hsl.

(A) Immunoblotting analysis of His6-tagged proteins in COS-7 cell extracts expressing His6-Hsl, His6-MmHsl, or His6-β-Gal. (B) Lipid hydrolase activities of recombinant Hsl. Cell extracts expressing …

Figure 2 with 1 supplement
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Normal TG and energy metabolism in Hsl1 mutant flies.

(A) Organization of the Hsl genomic region (including the neighboring genes Ate1 and PCNA), the Hsl gene locus and the Hsl1 deletion mutant allele. Black and white boxes represent coding and …

Figure 2—figure supplement 1
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Molecular characterization of the Hsl1 mutant allele.

(A) 1% agarose gel electrophoresis of diagnostic DNA products derived from genotyping PCRs of Hsl+ control and Hsl1 mutant genomic loci. DNA was amplified from fly homogenates using primers flanking …

Figure 3
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Sterol metabolism in Hsl1 mutant flies.

(A) SE levels in ad libitum fed control and Hsl1 mutant flies at different times after eclosion. Data are normalized to 1-day-old (1d) control animals (n = 6). (B) SE levels in Hslb24 mutant males …

Figure 4
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Adipocyte-autonomous control of organismal SE by Hsl.

(A) Distribution of SE in body segments and tissues of adult Hsl1 mutant and control animals (n = 3). (B) Lipid droplets in steroidogenic ring glands of Hsl1 mutant and control prepupae. (C) Whole …

Figure 5
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Maternal Hsl determines embryonic sterol homeostasis.

(A) Relative SE levels in embryos derived from reciprocal matings between Hsl1 (-/-) and control (+/+) animals at different times AEL (n = 8). (B) Free sterols in Hsl1 and control embryos at …

Figure 6 with 1 supplement
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Differentiation of the embryo lipidome in control and Hsl1 mutants.

(A) Total levels of neutral (left panel) and polar (right panel) lipid classes during embryogenesis of control and Hsl1 animals (n = 8). (B) Combined acyl chain lengths and double bonds in major …

Figure 6—figure supplement 1
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Changes in the lipidome during embryogenesis in control and Hsl1 mutants.

(A) Combined acyl chain lengths and double bonds in PG, PE O-, Cer, and CerPE (n = 8). Relative changes of individual glycerophospholipid species in control and Hsl1 animals between early (0–2 hr …

Figure 7 with 1 supplement
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Impaired fecundity and egg loading in Hsl1 mutant flies.

(A, B) Cumulative numbers of eggs and hatched 1st instar larvae per female on (A) LDM (n = 25–28) or (B) LDM with 0.01% cholesterol (n = 29). Note that LDM contains only traces of sterols and other …

Figure 7—figure supplement 1
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Hatching rates of control and Hsl1 mutant embryos.

Hatching rates of control and Hsl1 mutant embryos on (A) LDM (n = 25–28) and (B) LDM supplemented with 0.01% cholesterol (n = 29) were calculated from data depicted in Figure 7A,B. Data are …

Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Drosophila melanogaster)w1118Vienna Drosophila Research Center (VDRC)Cat#: 60000
Genetic reagent (Drosophila melanogaster)Hsl1This studySee Materials and methods
Genetic reagent (Drosophila melanogaster)UAS-HslThis studySee Materials and methods
Genetic reagent (Drosophila melanogaster)UAS-Hsl-EGFPThis studySee Materials and methods
Genetic reagent (Drosophila melanogaster)bmm1Grönke et al., 2005
doi: 10.1016/j.cmet.2005.04.003		FLYB: FBal0195572
Genetic reagent (Drosophila melanogaster)AkhAGáliková et al., 2015
doi: 10.1534/genetics.115.178897		FLYB: FBal0319563
Genetic reagent (Drosophila melanogaster)FB-GAL4Grönke et al., 2003
doi: 10.1016/s0960-9822(03)00175–1
Genetic reagent (Drosophila melanogaster)Act5c-GAL4Bloomington Drosophila Stock Center (BDSC)Cat#: 4414
FLYB: FBst0004414		backcrossed to a w1118 strain
Genetic reagent (Drosophila melanogaster)Cg-GAL4Bloomington Drosophila Stock Center (BDSC)Cat#: 7011
FLYB: FBst0007011
Genetic reagent (Drosophila melanogaster )nos-GAL4Bloomington Drosophila Stock Center (BDSC)Cat#: 64277
FLYB: FBst0064277
Genetic reagent (Drosophila melanogaster)Hsl shRNABloomington Drosophila Stock Center (BDSC)Cat#: 65148
FLYB: FBst0065148
Cell line (Chlorocebus aethiops)COS-7American Type Culture Collection (ATCC)Cat#: CRL-1651
RRID: CVCL_0224
AntibodyAnti-Mouse IgG-Horseradish Peroxidase antibody; sheepGE HealthcareCat#:NA931
RRID: AB_772210		WB
(1:5,000)
AntibodyAnti-Rabbit IgG (H+L)-Horseradish Peroxidase antibody; goat polyclonalVector LaboratoriesCat#: PI-1000
RRID: AB_2336198		WB
(1:5,000)
AntibodyAnti-engrailed/invected antibody; mouse monoclonalDevelopmental Studies Hybridoma Bank (DSHB)Cat#: 4D9
RRID: AB_528224		IHC
(1:100)
AntibodyHSL antibody; rabbit polyclonalCell Signaling TechnologyCat#: 4107
RRID: AB_2296900		WB
(1:1,000)
AntibodyRabbit Anti-GAPDH antibody, unconjugated, clone 14C10; rabbit monoclonalCell Signaling TechnologyCat#: 2118
RRID: AB_561053		WB
(1:10,000)
AntibodyAnti-His antibody, unconjugated; mouse monoclonalGE HealthcareCat#: 27-4710-01
RRID: AB_771435		WB
1:5000
Recombinant DNA reagentpFLC-1 [Hsl]Drosophila Genomics Resource Center (DGRC)Cat#: RE52776Used for amplification of Hsl cDNA
Transfected construct (D. melanogaster)pcDNA4/Hismax C [His6-Hsl]This studySee Materials and methods
Recombinant DNA reagentpUAST [Hsl]This studySee Materials and methods
Recombinant DNA reagentpUAST [Hsl-EGFP]This studySee Materials and methods
Sequence-based reagentOligonucleotides, PrimersThermo Fisher Scientific InvitrogenSee Materials and methods
Commercial assay or kitTriglycerides reagentThermo Fisher ScientificCat#: 981786
Commercial assay or kitNEFA-HR(2)Fujifilm Wako DiagnosticsCat#: 999–34691, Cat#: 995–34791 Cat#: 991–34891 Cat#: 993–35191 Cat#: 276–76491
Commercial assay or kitGlucose (GO) Assay kitSigma AldrichCat#: GAGO20-1KT
Commercial assay or kitRNeasy Mini kitQIAGENCat#: 74104
Commercial assay or kitQIAGEN Plasmid Midi KitQIAGENCat#: 12143
Commercial assay or kitTRIzolThermo Fisher Scientific InvitrogenCat#: 15596026
Commercial assay or kitSuperScript III First-Strand Synthesis SupermixThermo Fischer Scientific InvitrogenCat#: 18080051
Commercial assay or kitQuantiTect Reverse Transcription KitQIAGENCat#: 205313
Commercial assay or kitiTaq Universal SYBR Green SupermixBio-RadCat#: 1725120
Chemical compound, drugBODIPY 493/503Thermo Fisher Scientific InvitrogenCat#: B21035 µg/ml
Chemical compound, drugHoechst 33342Sigma AldrichCat#: 145335 µg/ml
Chemical compound, drugCellMask Deep RedThermo Fisher Scientific Life TechnologiesCat#: C100461 µg/ml
Chemical compound, drugLipidTOX Deep RedThermo Fisher Scientific Life TechnologiesCat#: H3477(1:1,000)
Chemical compound, drugLipid standardsAvanti Polar Lipids
Sigma Aldrich
Toronto Research Chemicals		See Materials and methods
Chemical compound, drugCholesterolSigma AldrichCat#: C3045
Chemical compound, drugCholesterol [25,26–3H]American Radiolabeled ChemicalsCat#: ART-1987
Software, algorithmLipidXplorer 1.2.7Herzog et al., 2012
doi:10.1371/journal.pone.0029851
Software, algorithmLipid Data AnalyzerHartler et al., 2011 doi:10.1093/bioinformatics/btq699
Table 1
Drosophila strains used in the study.
Name in text/figureFull genotypeID/Reference
Control or
+/+		w1118; +; +VDRC 60000
Hsl1 or
-/-		w1118; Hsl1; +This study
Hslb24w1118; Hslb24; +Bi et al., 2012
AkhAw1118; +; AkhAGáliková et al., 2015
bmm1w1118; +; bmm1Gáliková et al., 2017
AkhA bmm1w1118; +; AkhA bmm1This study
Hsl1 bmm1w1118; Hsl1; bmm1This study
Hsl1 AkhAw1118; Hsl1; AkhAThis study
UAS-Hsl or
+/+; UAS-Hsl		w1118; +; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+This study
Act5c>w*; P{w+mC=Act5C-GAL4}25FO1/+; +BDSC 4414
Act5c>UAS-Hslw*; P{w+mC=Act5C-GAL4}25FO1/+; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+This study, BDSC 4414
Hsl1 UAS-Hsl or
-/- UAS-Hsl		w1118; Hsl1; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+This study
Hsl1 Act5cw*; Hsl1 P{w+mC=Act5C-GAL4}25FO1/Hsl1; +This study, BDSC 4414
Hsl1 Act5c UAS-Hslw*; Hsl1 P{w+mC=Act5C-GAL4}25FO1/Hsl1; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+This study, BDSC 4414
FB>+ or
FB-GAL4		w*; P{w+mW.hs=GawB}FB/+; +Grönke et al., 2003
Hsl1 FBw*; Hsl1 P{w+mW.hs=GawB}FB/Hsl1; +This study
Hsl1 FB UAS-Hslw*; Hsl1 P{w+mW.hs=GawB}FB/Hsl1; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+This study
Hsl1 Myo31DFw*; Hsl1 P{w+mW.hs=GawB}Myo31DFNP0001/Hsl1; +This study, BDSC 67088
Hsl1 Myo31DF UAS-Hslw*; Hsl1 P{w+mW.hs=GawB}Myo31DFNP0001/Hsl1; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+This study, BDSC 67088
Hsl1 Desat1w*; Hsl1 P{w+mC=Desat1-GAL4.E800}2M/Hsl1; +This study, BDSC 65404
Hsl1 Desat1 UAS-Hslw*; Hsl1 P{w+mC=Desat1-GAL4.E800}2M/Hsl1; P{w+mC Hsl [Scer\UAS]=UAS-Hsl}/+This study, BDSC 65404
Hsl1 elavw*; Hsl1; P{w+mC=GAL4-elav.L}3/+This study, BDSC 8760
Hsl1 elav UAS-Hslw*; Hsl1; P{w+mC=GAL4-elav.L}3/ P{w+mC Hsl [Scer\UAS]=UAS-Hsl}This study, BDSC 8760
-/-; nos-GAL4w*; Hsl1; P{w+mC=GAL4::VP16-nos.UTR}1C/+This study, BDSC 64277
-/-; nos-GAL4/UAS-Hslw*; Hsl1; P{w+mC=GAL4::VP16-nos.UTR}1C/ P{w+mC Hsl [Scer\UAS]=UAS-Hsl}This study, BDSC 64277
Cg>+w[1118]/y[1] v[1]; P{w[+mC]=Cg-GAL4.A}2/ P{y[+t7.7]=CaryP}attP40; +BDSC 7011 and BDSC 36304
Hsl shRNA/+w[1118]/y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMC05951}attP40/+; +BDSC 65148 and VDRC 60000
Cg>Hsl shRNAw[1118]/y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMC05951}attP40/ P{w[+mC]=Cg-GAL4.A}2; +BDSC 7011 and BDSC 65148
Table 2
List of primers used for RT-qPCR.
Gene symbolPrimer sequencesID/Reference
 rp49/RpL32fw: 5‘-CTTCATCCGCCACCAGTC-3‘ rv: 5‘-CGACGCACTCTGTTGTCG-3‘
 plin1fw: 5‘-GCGCGAATTCTGGCGCCCCTAGATG-3‘ rv: 5‘-CACAGAAGTAAGGTTCCTTCACAAAGATCC-3‘
 plin2fw: 5’-TCAAATTGCCCGTGGTAAA-3’ rv: 5’-CCCATTCGAAGACACGATTT-3’
 Akh-QIAGEN QuantiTect
QT00957859
 bmm-QIAGEN QuantiTect
QT00964460
 Hr96fw: 5’-CCAGCGAGGCTCTTTATGAT-3’ rv: 5’-GGTTGTGGCGAGTGTCGT-3’
 Npc1afw: 5’-GTCGAGGAACTTTGCAGGGA-3’ rv: 5’-TCATCGAAACAGGACTGCGT-3’
 Npc1bfw: 5’-CGGATTTTGTTCCAGCAACT-3’ rv: 5’-CCATTCTCAGTAAATCCTCGTTC-3’
 Npc2afw: 5’-ACAGTCGTCCACGGCAAG-3’ fw: 5’-ACACAGGCATCGGGATTG-3’
 Npc2bfw: 5‘-GGAGATCCACTGGGGATTG-3‘ rv: 5‘-CCTTGATTTTGGCGGGTAT-3‘
 CG8112fw: 5’-CACAAACTGAAACCGCACAG-3’ rv: 5’-CGACACGAAACAGAAGACCA-3’
 magrofw: 5’-ACACCGAACTGATTCCGAAC-3’ rv: 5’-ATCCACCATTGGCAAACATT-3’
 Hslfw: 5‘-CTGGAGGCGACCTATGGAAC-3‘ rv: 5‘-GCTCGTCAAAATCGTACTCGTG-3‘
 PCNAfw: 5‘-GCGACCGCAATCTCTCCAT-3‘ rv: 5‘-CGCCTTCATCGTCACATTGT-3‘
 Ate1fw: 5‘-GCATACTTCGCCGCATAAATCG-3‘ rv: 5‘-CTATGGCGTAATCGGCATCGG-3‘
 MmAbca1fw: 5’-GATGTGGAATCGTCCCTCAGTTC-3’ rv: 5’-ACTGCTCTGAGAAACACTGTCCTCC-3’
 MmSoat1fw: 5’-GAAACCGGCTGTCAAAATCTGGR-3’ rv: 5’-TGTGACCATTTCTGTATGTGTCC-3’
 MmHmgcs1fw: 5’-GACAAGAAGCCTGCTGCCATA-3’ rv: 5’-CGGCTTCACAAACCACAGTCT-3’
 MmHmgcrfw: 5’-TGCACGGATCGTGAAGACA-3’ rv: 5’-GTCTCTCCATCAGTTTCTGAACCA-3’
 MmSrebp2fw: 5’-GCGCCAGGAGAACATGGT-3’ rv: 5’-CGATGCCCTTCAGGAGCTT-3’
 MmStarfw: 5’-TTGGGCATACTCAACAACCA-3’ rv: 5’-GAAACACCTTGCCCACATCT-3’
 Mm36B4fw: 5’-GCTTCATTGTGGGAGCAGACA-3’ rv: 5’-CATGGTGTTCTTGCCCATCAG-3’

Additional files

Supplementary file 1

Composition of the lipidome.

https://cdn.elifesciences.org/articles/63252/elife-63252-supp1-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/63252/elife-63252-transrepform-v2.docx

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