Enhanced C/EBPβ function promotes hypertrophic versus hyperplastic fat tissue growth and prevents steatosis in response to high-fat diet feeding

  1. Christine Müller
  2. Laura M Zidek
  3. Sabrina Eichwald
  4. Gertrud Kortman
  5. Mirjam H Koster
  6. Cornelis F Calkhoven  Is a corresponding author
  1. European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Netherlands
  2. Leibniz Institute on Aging - Fritz Lipmann Institute, Germany
  3. Division Molecular Genetics, Department of Pediatrics, University Medical Center Groningen, University of Groningen, Netherlands
8 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
CebpbΔuORF mice on high-fat diet (HFD).

(A) Growth curves of wt and CebpbΔuORF (ΔuORF) male mice on HFD (wt, n = 10; CebpbΔuORF, n = 8). (B) Volume of total fat mass as measured by abdominal CT analyses (males,19 weeks; ND, n = 5; HFD, n = 4). (C) Volume of visceral fat mass as measured by abdominal CT analyses (males,19 weeks; ND, n = 5; HFD, n = 4) (D) Volume of subcutaneous fat mass as measured by abdominal CT analyses (males, 19 weeks; ND, n = 5; HFD, n = 4). (E) Female body weight (week 19; ND wt, n = 7; HFD wt, n = 4; ND and HFD ΔuORF, n = 6). (F) Visceral fat weight (females, week 19; ND wt, n = 7; HFD wt, n = 4; ND and HFD ΔuORF, n = 6). (G) Subcutaneous fat weight (females, week 19; ND wt, n = 7; HFD wt, n = 4; ND and HFD ΔuORF, n = 6). All values are mean ± SEM. p-Values were determined with Student’s t-test, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 1—figure supplement 1
Food intake, energy efficiency and lean mass of male mice on high-fat diet (HFD).

(A) Daily food intake per mouse on HFD, normalized to body weight as determined over 18 weeks (males, wt, n = 9 mice / 4 cages; ΔuORF n = 7 mice / 2 cages). (B) Efficiency of caloric utilization in males on HFD measured by bomb calorimetry of food and feces (wt, n = 4; ΔuORF n = 4). (C) Volume of lean body mass of CebpbΔuORF male mice measured by abdominal CT analyses (19 weeks; ND, n = 5; HFD n = 4). All values are mean ± SEM. P-values were determined with Student’s t-test, *p < 0.05.

CebpbΔuORF mice on high-fat diet (HFD) store fat in hyperplastic adipocytes.

Histological hematoxylin and eosin (H&E) staining of epididymal WAT from (A) males (19 weeks HFD) and (B) females (19 weeks HFD). Quantification of the fat cell area is shown at the right (males: wt, n = 7; ΔuORF, n = 4; females: wt, n = 4; ΔuORF, n = 7; 12 adjacent cells are measured per mouse).

Inflammation of the visceral WAT is reduced in CebpbΔuORF male mice on high-fat diet (HFD).

(A) Relative mRNA expression levels of the macrophage marker CD68 measured in the visceral fat of CebpbΔuORF (ΔuORF) and wt male mice on either normal diet (ND) or HFD (19 weeks; ND, n = 5; HFD, n = 4). (B) Immunohistological staining of the visceral fat of CebpbΔuORF male mice (ΔuORF) and wt mice on HFD (19 weeks) using a CD68-specific antibody (arrow points to specific staining). Histological sections from three individual mice per genotype are shown. (C) Relative mRNA expression levels of the inflammatory cytokines TNFα, MCP1, IL-1β, and IL6 measured in the visceral fat of CebpbΔuORF (ΔuORF) and wt male mice on either normal diet (ND) or HFD (19 weeks; wt: ND, n = 5; HFD, n = 6; ΔuORF: ND, n = 6 (for IL-6, n = 4, the results of two mice were excluded due to undetectable signal); HFD, n = 4). All values are mean ± SEM. p-Values were determined with Student’s t-test, *p < 0.05; ***p < 0.001.

Macrophage infiltration of the visceral WAT is reduced in CebpbΔuORF female mice on high-fat diet (HFD).

(A) Relative mRNA expression levels of the macrophage marker CD68 measured in the visceral fat of CebpbΔuORF (ΔuORF) and wt female mice on either normal diet (ND) or HFD (19 weeks; wt: ND, n = 7; HFD, n = 4; ΔuORF: ND, n = 4 (the result from one mouse was excluded due to undetectable signal); HFD, n = 5). (B) Immunohistological staining of the visceral fat of CebpbΔuORF female mice (ΔuORF) and wt mice on HFD (19 weeks) using a CD68-specific antibody (arrow points to specific staining). Histological sections from three individual mice per genotype are shown. (C) Relative mRNA expression levels of the inflammatory cytokines TNFα, MCP1, IL-1β, and IL6 measured in the visceral fat of CebpbΔuORF female mice (ΔuORF) and wt mice on either normal diet (ND) or HFD (19 weeks; wt: ND, n = 6; HFD, n = 4; ΔuORF: ND, n = 5; HFD, n = 5). All values are mean ± SEM. p-Values were determined with Student’s t-test, *p < 0.05; **p < 0.01.

Figure 5 with 1 supplement
CebpbΔuORF mice on high-fat diet (HFD) are protected against steatosis.

Histological sections of liver from (A) males and (B) females of wt or CebpbΔuORF mice (ΔuORF) (19 weeks). Sections were stained with hematoxylin (blue) and Sudan III (males) or Oil-Red-O (females) for red color lipid staining. Liver weight of (C) males and (D) females of wt or CebpbΔuORF mice (ΔuORF) (19 weeks; males: ND, n = 6; HFD, n = 4; females: wt ND, n = 7, wt HFD, n = 4; ΔuORF wt and HFD, n = 6). All values are mean ± SEM. p-Values were determined with Student’s t-test, *p < 0.05; **p < 0.01.

Figure 5—figure supplement 1
CebpbΔuORF mice on high-fat diet (HFD) are protected against steatosis in the heart and skeletal muscle.

(A) Histological sections of cardiac muscle and (B) skeletal muscle of wt or CebpbΔuORF male mice (ΔuORF) (19 weeks). Sections were stained with hematoxylin (blue) and Sudan III for red color lipid staining. (C) Heart weights of CebpbΔuORF and wt males and (D) females as indicated on normal diet (ND) or HFD (19 weeks, males: wt ND, n = 4; wt HFD, n = 3, ΔuORF ND and HFD, n = 4; females: wt ND, n = 7, wt HFD, n = 4, ΔuORF ND and HFD, n = 6). All values are mean ± SEM. p-Values were determined with Student’s t-test, *p < 0.05; **p < 0.01.

CebpbΔuORF mice show improved glucose tolerance and insulin sensitivity on a high-fat diet (HFD).

Intraperitoneal glucose tolerance test (IPGTT) with the calculated area under the curve (AUC) of CebpbΔuORF (A) male and (B) female (ΔuORF) and wt mice injected i.p. with glucose (2 g/kg) after a 16 hr fast (7 weeks; males: wt ND, n = 6; wt HFD, n = 9; ΔuORF ND, n = 6; ΔuORF HFD, n = 7; females: wt ND, n = 7; wt HFD, n = 6; ΔuORF ND and HFD, n = 6). Intraperitoneal insulin sensitivity test (IPIST) with the calculated area under the curve (AUC) of CebpbΔuORF (C) male and (D) female (ΔuORF) mice and wt mice injected i.p. with insulin (0.5 IU/kg) (7 weeks; males: wt ND, n = 6; wt HFD, n = 8; ΔuORF ND, n = 5; ΔuORF HFD, n = 7; females: wt ND, n = 7; wt HFD, n = 7; ΔuORF ND, n = 6; ΔuORF HFD, n = 7). All values are mean ± SEM. p-Values were determined with Student’s t-test, *p < 0.05; ***p < 0.001.

Figure 7 with 1 supplement
Expression of key adipogenic genes is elevated in CebpbΔuORF male mice on high-fat diet (HFD).

Relative mRNA expression levels of the adipogenic transcription factors C/EBPα, PPARγ and SREBBP1c and key enzyme FAS measured visceral WAT of CebpbΔuORF (A) male and (B) female (ΔuORF) mice and wt mice on either normal diet (ND) or HFD (19 weeks; males: wt ND, n = 5; wt HFD, n = 7; ΔuORF ND, n = 6; ΔuORF HFD, n = 4; females: wt ND, n = 6; wt HFD, n = 4; ΔuORF ND and HFD, n = 5). All values are mean ± SEM. p-Values were determined with Student’s t-test, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7—figure supplement 1
Protein expression of key adipogenic genes is elevated in CebpbΔuORF mice on high-fat diet (HFD).

(A) Immunoblot of the adipogenic transcription factors C/EBPα, PPARγ and SREBP1 and the key enzyme FAS performed with visceral WAT extracts from CebpbΔuORF male mice (ΔuORF) and wt mice on either normal diet (ND) or HFD (19 weeks). An immunoblot of GAPDH served as loading control. (B) Immunoblot of the key enzyme FAS performed with liver extracts from CebpbΔuORF female mice (ΔuORF) and wt mice on either normal diet (ND) or HFD (19 weeks). An immunoblot of GAPDH served as loading control.

LAP and LIP expression under ND and HFD feeding.

(A) Immunoblots of C/EBPβ and GAPDH loading control performed with visceral WAT extracts from wt or CebpbΔuORF males on either normal diet (ND) or HFD (19 weeks). (B) Quantification of the LAP/LIP ratio in split bar diagrams for better visualization, and (C) quantification of LAP and LIP isoform expression separately (normalized to the GAPDH signal) using the whole cohort (wt ND, n = 5; wt HFD, n = 6; ΔuORF ND, n = 6; ΔuORF HFD, n = 5). (D) Immunoblots of C/EBPβ and GAPDH loading control performed with visceral WAT extracts from wt or CebpbΔuORF females on either normal diet (ND) or HFD (19 weeks). (E) Quantification of the LAP/LIP ratios in split bar diagrams for better visualization, and (F) quantification of LAP and LIP isoform expression separately (normalized to the GAPDH signal) using the whole cohort (wt ND, n = 8; wt HFD, n = 4; ΔuORF ND and HFD, n = 6). All values are mean ± SEM. p-Values were determined with Student’s t-test, *p < 0.05; **p < 0.01; ***p < 0.001.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (M. musculus)C/EBPβΔuORFhttps://doi.org.10.1101/gad.557910https://doi.org.10.15252/embr.201439837males, back-crossed for 6 generations and females, back-crossed for 12 generations into C57BL/6 J background
AntibodyAnti-C/EBPβ (E299) (rabbit monoclonal)AbcamCat# ab32358, RRID:AB_726796(1:1000)
AntibodyAnti-C/EBPα (D56F10) (rabbit monoclonal)Cell SignalingCat# 8178, RRID:AB_11178517(1:1000)
AntibodyAnti-PPARγ (C26H12) (rabbit monoclonal)Cell SignalingCat# 2435, RRID:AB_2166051(1:1000)
AntibodyAnti-FAS (C20G5) (rabbit monoclonal)Cell SignalingCat# 3180, RRID:AB_2100796(1:1000)
AntibodyAnti-GAPDH (14 C10) (rabbit monoclonal)Cell SignalingCat# 2118, RRID:AB_561053(1:1000)
AntibodyAnti-SREBP1 (2 A4) (mouse monoclonal)NeoMarkersCat# MS-1207-PO(1:1000)
AntibodyAnti-CD68 (E307V) (rabbit monoclonal)Cell SignalingCat# 97,778(1:200)
AntibodyAnti-rabbit IgG, HRP-conjugated (donkey polyclonal)GE HealthcareCat#: NA934, RRID:AB_772206(1:5000)
AntibodyAnti-mouse IgG, HRP-conjugated (sheep polyclonal)GE HealthcareCat#: NXA931, RRID:AB_772209(1:5000)
AntibodyAnti-rabbit IgG, biotin-conjugated (goat polyclonal)Vector LabsCat#: BA-1000(1:250)
Sequence-based reagentCD68 (F)https://doi.org.10.7554/eLife.34985.001PCR primer5’-GCCCACCAC CACCAGTCACG –3’
Sequence-based reagentCD68 (R)https://doi.org.10.7554/eLife.34985.001PCR primer5’GTGGTCCAG GGTGAGGGCC A-3’
Sequence-based reagentPPARγ (F)https://doi.org.10.15252/embr.201439837PCR primer5’-GCCCTTTGG TGACTTTATGG –3’
Sequence-based reagentPPARγ (R)https://doi.org.10.15252/embr.201439837PCR primer5’-CAGCAGGTT GTCTTGGATGT 3’
Sequence-based reagentC/EBPα (F)https://doi.org.10.15252/embr.201439837PCR primer5’-CAAGAACAG CAACGAGTACC G-3’
Sequence-based reagentC/EBPα (R)https://doi.org.10.15252/embr.201439837PCR primer5’-GTCACTGGT CAACTCCAGCA C-3’
Sequence-based reagentSREBP1c (F)https://doi.org.10.15252/embr.201439837PCR primer5’-AACGTCACT TCCAGCTAGAC –3’
Sequence-based reagentSREBP1c (R)https://doi.org.10.15252/embr.201439837PCR primer5’-CCACTAAGG TGCCTACAGAG C-3’
Sequence-based reagentFAS (F)https://doi.org.10.15252/embr.201439837PCR primer5’-ACACAGCAA GGTGCTGGAG-3’
Sequence-based reagentFAS (R)https://doi.org.10.15252/embr.201439837PCR primer5’-GTCCAGGCT GTGGTGACTCT –3’
Sequence-based reagentTNFα (F)This paperPCR primer5’-CCAGACCCT CACACTCA-3’
Sequence-based reagentTNFα (R)This paperPCR primer5’-CACTTGGTG GTTTGCTACGA C-3’
Sequence-based reagentMCP1 (F)This paperPCR primer5‘-GCTGGAGAG CTACAAGAGGA TCA-3’
Sequence-based reagentMCP1 (R)This paperPCR primer5‘-ACAGACCTC TCTCTTGAGCT TGGT-3
Sequence-based reagentIL-1β (F)This paperPCR primer5‘-GAAATGCCA CCTTTTGACAG TG-3’
Sequence-based reagentIL-1β (R)This paperPCR primer5‘-TGGATGCTC TCATCAGGACA G-3
Sequence-based reagentIL-6 (F)This paperPCR primer5’-CCGGAGAGG AGACTTCACAG –3’
Sequence-based reagentIL-6 (R)This paperPCR primer5’-TTCTGCAAG TGCATCATCGT –3’
Sequence-based reagentGAPDH (F)This paperPCR primer5’-ATTGTCAGC AATGCATCCTG –3’
Sequence-based reagentGAPDH (R)This paperPCR primer5’-ATGGACTGT GGTCATGAGC C-3’
Sequence-based reagentβ-actin (F)https://doi.org.10.15252/embr.201439837PCR primer5’-AGAGGGAAA TCGTGCGTGA C-3'
Sequence-based reagentβ-actin (R)https://doi.org.10.15252/embr.201439837PCR primer5'-CAATAGTGA TGACCTGGCC GT-3’
Commercial assay or kitVectastain ABC HRP KitVector LabsCat#: PK-4000
Commercial assay or kitWestern Lightning Plus ECL ReagentPerkin EmerCat#: NEL103001EA
Commercial assay or kitECL Prime Western Blotting ReagentGE HealthcareCat#: RPN2236
Commercial assay or kitRestore Western Blot Stripping bufferThermo FisherCat#: 21,063
Commercial assay or kitQIAzol Lysis re-agentQIAGENCat#: ID:79,306
Commercial assay or kitRNeasy Lipid Tissue Mini kitQIAGENCat#: ID:74,804
Commercial assay or kitRneasy Plus Mini kitQIAGENCat#: ID:74,134
Commercial assay or kitTranscriptor First Strand cDNA Synthesis kitRocheCat#: 4379012001
Commercial assay or kitLight Cycler 480 SYBR Green I Master MixRocheCat#: 0470751600
Chemical compound, drugInsulin (human)LillyCat#: HI-210
Chemical compound, drugSudan IIISigma-AldrichCat#: S4136
Chemical compound, drugOil-Red-OSigma-AldrichCat#: O0625
Software, algorithmGraphPad Prism 9.0Graphpad Software, La Jolla, CARRID:SCR_002798
Software, algorithmImage Quant LAS 4000 Mini Imager SoftwareGE HealthcareRRID:SCR_014246
Software, algorithmImageJhttps://doi.org.10.1186/s12859-017-1934-zRRID:SCR_003070

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  1. Christine Müller
  2. Laura M Zidek
  3. Sabrina Eichwald
  4. Gertrud Kortman
  5. Mirjam H Koster
  6. Cornelis F Calkhoven
(2022)
Enhanced C/EBPβ function promotes hypertrophic versus hyperplastic fat tissue growth and prevents steatosis in response to high-fat diet feeding
eLife 11:e62625.
https://doi.org/10.7554/eLife.62625