Health benefits attributed to 17α-estradiol, a lifespan-extending compound, are mediated through estrogen receptor α

  1. Shivani N Mann
  2. Niran Hadad
  3. Molly Nelson Holte
  4. Alicia R Rothman
  5. Roshini Sathiaseelan
  6. Samim Ali Mondal
  7. Martin-Paul Agbaga
  8. Archana Unnikrishnan
  9. Malayannan Subramaniam
  10. John Hawse
  11. Derek M Huffman
  12. Willard M Freeman
  13. Michael B Stout  Is a corresponding author
  1. Department of Nutritional Sciences, University of Oklahoma Health Sciences Center, United States
  2. Oklahoma Center for Geroscience, University of Oklahoma Health Sciences Center, United States
  3. Harold Hamm Diabetes Center, University of Oklahoma Health Sciences Center, United States
  4. The Jackson Laboratory, United States
  5. Department of Biochemistry and Molecular Biology, Mayo Clinic, United States
  6. Department of Cell Biology, University of Oklahoma Health Sciences Center, United States
  7. Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, United States
  8. Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, United States
  9. Department of Molecular Pharmacology, Albert Einstein College of Medicine, United States
  10. Genes & Human Disease Research Program, Oklahoma Medical Research Foundation, United States
  11. Oklahoma City Veterans Affairs Medical Center, United States
6 figures, 1 table and 4 additional files

Figures

Figure 1 with 1 supplement
17α-E2 and 17β-E2 elicit similar genomic binding and transcriptional profiles through ERα.

(A) Heatmap representing normalized genome-wide DNA binding by ERα via ChIP sequencing analyses centered according to peak summits for each treatment group and compared to each other group. (B) …

Figure 1—figure supplement 1
17α-E2 and 17β-E2 elicit similar ERα binding profile.

PCA plot of genome-wide ERα binding profiles by ChIP sequencing analyses. U2OS-ERα cells treated with low dose (10 nM) 17α-E2, high dose (100 nM) 17α-E2, or 17β-E2 (10 nM) induced binding in common …

ERα is required for 17α-E2 to reduce mass, adiposity, and calorie intake in male mice.

(A) Percent change in mass (mean ± SEM, two-way repeated measures ANOVA with Holm-Sidak post-hoc; *p<0.05, **p<0.005 between WT HFD and WT HFD+17α), (B) Mass at baseline (week 0; solid) and week 14 …

Figure 3 with 2 supplements
17α-E2 reverses obesity-related metabolic dysfunction in male WT, but not ERα KO, mice.

(A) Fasting glucose (mean ± SEM, two-way repeated measures ANOVA), (B) Fasting insulin (mean ± SEM, two-way repeated measures ANOVA with Holm-Sidak post-hoc; *p<0.05, **p<0.005), and (C) …

Figure 3—figure supplement 1
17α-E2 reverses obesity-related metabolic dysfunction in male WT, but not ERα KO, mice.

(A) Glucose tolerance testing (GTT; 1 mg/kg), normalized to baseline (min 0) (mean ± SEM, two-way repeated measures ANOVA with Holm-Sidak post-hoc; *p<0.05 between WT HFD and WT HFD+17α), and (B) …

Figure 3—figure supplement 2
17α-E2 fails to alter metabolic parameters in WT or ERα KO female mice.

(A) Mass at baseline (week 0; solid) and week 4 (striped) (mean ± SEM, two-way repeated measures ANOVA), (B) Fat mass at baseline (week 0; solid) and week 4 (striped) (mean ± SEM, two-way repeated …

Figure 4 with 2 supplements
17α-E2 reverses obesity-related hepatic steatosis in an ERα-dependent manner in male mice.

(A) Liver mass (mean ± SEM, two-way ANOVA with Holm-Sidak post-hoc; *p<0.05), (B) Representative liver oil-red-O staining, (C) Liver fatty acids (mean ± SEM, two-way ANOVA with Holm-Sidak post-hoc; …

Figure 4—figure supplement 1
17α-E2 alters markers of lipid and glucose homeostasis predominantly through ERα in male mice.

(A) Quantification of oil-red-O lipid staining in liver sections from WT and ERα KO mice provided 45% HFD (TestDiet 58V8)±17α-E2 (14.4ppm) for 14 weeks (mean ± SEM, two-way ANOVA with Holm-Sidak …

Figure 4—figure supplement 2
17α-E2 alters the hepatic fatty acid profile in male WT, but not ERα KO, mice.

Relative molar % of fatty acids in the liver from WT and ERα KO mice provided 45% HFD (TestDiet 58V8)±17α-E2 (14.4ppm) for 14 weeks (mean ± SEM, Benjamini–Hochberg multiple testing correction, …

17α-E2 reverses obesity-related liver fibrosis and insulin resistance in an ERα-dependent manner in male mice.

(A) Representative liver Masson’s trichrome staining for collagen and (B) Liver transcriptional markers of fibrosis in WT and ERα KO mice provided 45% HFD (TestDiet 58V8)±17α-E2 (14.4ppm) for 14 …

Acute delivery of 17α-E2 improves hepatic insulin sensitivity.

(A) Schematic of peripheral 17α-E2 infusions (or vehicle) during hyperinsulinemic-euglycemic clamps, (B) glucose infusion rates (GIR) (mean ± SEM, unpaired Student’s t-test; **p<0.005), (C) rate of …

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (M. musculus)B6N(Cg)-Esr1tm4.2Ksk/JThe Jackson LaboratoryStock No:026176;
RRID:IMSR_JAX:026176
ERα (Esr1) KO mice
Cell line (Homo sapien)U2OS CellsATCCHTB-96;
RRID:CVCL_0042
PMID:15802376
PMID:14505348
Antibodyanti-FLAG M2 (Mouse monoclonal)Sigma-AldrichF1804IP: 1 uL per pull-down (1 mg/mL)
Commercial assay or kitProtein G DynabeadsApplied Biosystems/Thermofisher Scientific10003DIP: 30 uL per IP
Chemical compound, drug17α-E2Steraloids, IncE0870-000
Chemical compound, drugNovolin R 100 U/mlNovolin2mU/g
Other (diet)Chow; TestDiet 58YPTestDietTestDiet 58YP
Other (diet)HFD; TestDiet 58V8TestDietTestDiet 58V8HFD 45% by kcal
Other (diet)HFD; TestDiet 58Y1TestDietTestDiet 58Y1HFD 60% by kcal
Commercial assay or kitMouse Ultrasensitive Insulin ELISAALPCOCat# 80-INSMSU-E01; RRID:AB_2792981
Commercial assay or kitFree Glycerol AgentSigma-AldrichSigma F6428
Commercial assay or kitTriglyceride ReagentSigma-AldrichSigma F6428
Commercial assay or kitGlycerol StandardSigma-AldrichSigma G1394
Antibodyanti-pS473 AKT (Rabbit polyclonal)AbcamCat# ab81283; RRID:AB_2224551WB: (1:3000)
AntibodyAnti-pan-AKT (Rabbit polyclonal)AbcamCat# ab179463; RRID:AB_2810977WB (1:10000)
AntibodyAnti-pS256 FOX01 (Rabbit polyclonal)AbcamCat# ab131339; RRID:AB_11159015WB (1:1000)
AntibodyAnti-FOX01a (Rabbit polyclonal)AbcamCat# ab52857; RRID:AB_869817WB (1:1000)
AntibodyAnti-GAPDH (Rabbit polyclonal)AbcamCat# ab9485; RRID:AB_307275WB (1:2500)
AntibodyAnti-Rabbit IgG, IRDye 800 CWLI-CORCat# 926–32211; RRID:AB_621843WB (1:15000)
Commercial assay or kitTaqMan Gene Expression Master MixApplied Biosystems/Thermofisher Scientific4369542
Sequenced-based reagentqPCR primer Mmp1Integrated DNA TechnologiesMm.PT.58.42286812
Ref Seq: NM_008607(1)
Exon 5–6
Sequenced-based reagentqPCR primer Mmp12Integrated DNA TechnologiesMm.PT.58.31615472
Ref Seq: NM_008605(1)
Exon 8–9
Sequenced-based reagentqPCR primer Ccl2Integrated DNA TechnologiesMm.PT.58.42151692
Ref Seq: NM_011333(1)
Exon 1–3
Sequenced-based reagentqPCR primer Srebf1Integrated DNA TechnologiesMm.PT.58.8508227
Ref Seq: NM_011480(1)
Exon 1–2
Sequenced-based reagentqPCR primer Pck1Integrated DNA TechnologiesMm.PT.58.11992693
Ref Seq: NM_011044(1)
Exon 3–4
Sequenced-based reagentqPCR primer Cdkn1aIntegrated DNA TechnologiesMm.PT.58.17125846
Ref Seq: NM_007669(1)
Exon 2–3
Sequenced-based reagentqPCR primer PparαIntegrated DNA TechnologiesMm.PT.58.9374886
Ref Seq: NM_001113418(2)
Exon 8–9
Sequenced-based reagentqPCR primer Cxcl1Integrated DNA TechnologiesMm.PT.58.42076891
Ref Seq: NM_008176(1)
Exon 2–4
Sequenced-based reagentqPCR primer Col1a1Integrated DNA TechnologiesMm.PT.58.7562513
Ref Seq: M_007742(1)
Exon 1–2
Sequenced-based reagentqPCR primer Tnfrsf1aIntegrated DNA TechnologiesMm.PT.58.28810479
Ref Seq: NM_011609(1)
Exon 5–7
Software, algorithmSigmaPlot 12.5Systat SoftwareRRID:SCR_003210statistical analyses
Software, algorithmImageJImageJRRID:SCR_003070histological quantification
Software, algorithmImage StudioLI-CORRRID:SCR_015795western blot quantification
Software, algorithmRStudioGenomicAlignments
DiffBind
DESeq2
GenomicRanges
RRID:SCR_000432Peak Calling
Differential expression
Differential binding
Software, algorithmBowtie2
MACS2
Bedtools
Samtools
Picard-tools
Trimmomatic
Bowtie2
MACS2
Bedtools
Samtools
Picard-tools
Trimmomatic
Alignment, Peak Calling, trimming, duplicate identification

Additional files

Supplementary file 1

Pairwise statistical comparisons of ERα binding.

Negative binomial regression Wald post-hoc comparison test, FDR < 0.05. n = 3/group.

https://cdn.elifesciences.org/articles/59616/elife-59616-supp1-v2.xlsx
Supplementary file 2

ERα binding motif analysis.

Motif analysis was performed using HOMER with standard settings with the significance threshold set to FDR corrected p<0.05. Peak regions called for each treatment group were analyzed to identify enriched motifs relative to the entire genome. Hypergeometric test was used to test enrichment. Only motifs with FDR corrected p<0.05 were reported as significant. For pairwise differential motif enrichment or depletion across experimental groups, we utilized the hypergeometric test by using the number of sequences with motif from each group and total number of peaks as total sample size. Motifs that appear in less than five sequences between both test groups were removed. Benjamini-Hochberg multiple testing correction was utilized to control for false discovery rate (FDR < 0.05).

https://cdn.elifesciences.org/articles/59616/elife-59616-supp2-v2.xlsx
Supplementary file 3

Circulating eicosanoid levels (pmol/ml).

17α-E2 mildly alters the circulating eicosanoid profile in obese middle-aged male mice. WT mice were provided 45% HFD (TestDiet 58V8)±17α-E2 (14.4ppm) for 14 weeks. Age-matched, male WT, chow-fed (TestDiet 58YP) mice were also evaluated as a normal-weight reference group. All data are presented as mean ± SEM and were analyzed by Student’s t-test with the WT Chow group being excluded from statistical comparisons. n = 5–7.

https://cdn.elifesciences.org/articles/59616/elife-59616-supp3-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/59616/elife-59616-transrepform-v2.docx

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