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Distinct regulatory mechanisms governing embryonic versus adult adipocyte maturation

Nature Cell Biology volume 17, pages 1099–1111 (2015)Cite this article

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Abstract

Pathological expansion of adipose tissue contributes to the metabolic syndrome. Distinct depots develop at various times under different physiological conditions. The transcriptional cascade mediating adipogenesis is established in vitro, and centres around a core program involving PPARγ and C/EBPα. We developed an inducible, adipocyte-specific knockout system to probe the requirement of key adipogenic transcription factors at various stages of adipogenesis in vivo. C/EBPα is essential for all white adipogenic conditions in the adult stage, such as adipose tissue regeneration, adipogenesis in muscle and unhealthy expansion of white adipose tissue during high-fat feeding or due to leptin deficiency. Surprisingly, terminal embryonic adipogenesis is fully C/EBPα independent, but does however depend on PPARγ; cold-induced beige adipogenesis is also C/EBPα independent. Moreover, C/EBPα is not vital for adipocyte survival in the adult stage. We reveal a surprising diversity of transcriptional signals required at different stages of adipogenesis in vivo.

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Figure 1: C/EBPα is not required for terminal embryonic adipogenesis.
Figure 2: C/EBPα-deficient adipocytes are viable and have normal lipid accumulation in vitro.
Figure 3: Normal viability and lipid accumulation in inducible C/EBPα-deficient adipocytes in vivo.
Figure 4: Mice with inducible C/EBPα elimination in adipocytes have impaired glucose and lipid metabolism during HFD challenge.
Figure 5: Inducible deletion of C/EBPα in adipocytes of ob/ob mice attenuates adipose tissue expansion, which can be rescued by adiponectin overexpression.
Figure 6: C/EBPα is required for terminal white adipogenesis in the adult stage.
Figure 7: Terminal beige adipogenesis does not depend on C/EBPα.
Figure 8: The complexity of adipogenesis in vivo.

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Acknowledgements

We thank the UT Southwestern Histology Core for assistance in embedding and processing of tissue samples. We would also like to thank Shimadzu Scientific Instruments for the collaborative effort and advice on the mass spectrometric instrumentation side. This study was supported by US National Institutes of Health (NIH) grants R01-DK55758, P01-DK088761 and R01-DK099110 (P.E.S.). Q.A.W. is supported by a postdoctoral fellowship from the American Diabetes Association (7-11-MN-47). R.K.G. is supported by NIH grants R01-DK104789 and R03-DK099428 and the Searle Scholars Program (Chicago, IL).

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Authors and Affiliations

  1. Department of Internal Medicine, Touchstone Diabetes Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    Qiong A. Wang, Caroline Tao, Mengle Shao, Risheng Ye, Yi Zhu, Ruth Gordillo, Aktar Ali, William L. Holland, Rana K. Gupta & Philipp E. Scherer

  2. Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    Lei Jiang

  3. Division of Eli Lilly and Company, LIFA Diabetes, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA

    Yi Zhu

  4. Center for Immunology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    Yun Lian

  5. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    Philipp E. Scherer

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Contributions

Q.A.W., R.K.G. and P.E.S. designed the experiments and wrote the manuscript. Q.A.W., C.T., L.J., M.S., R.Y., Y.Z., R.G., A.A. and R.K.G. performed experiments and analysed data. Y. L. and W.L.H. analysed data.

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Correspondence to Philipp E. Scherer.

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Integrated supplementary information

Supplementary Figure 1 Adipocyte specific inducible knockout of C/EBPα in maturing and mature adipocytes.

a. Inducible, mature adipocytes specific knockout of C/EBPα. In the Adn-C/EBPαflox/flox mice, C/EBPα is expressed normally in every cell. On doxycycline treatment, rtTA will activate the TRE promoter to induce Cre expression. Cre protein will subsequently cut the floxed region in C/EBPα and eliminate the expression of C/EBPα in all existing mature adipocytes (Adn-C/EBPα−/−). Mice lacking TRE–Cre or Adn-rtTA were used as controls. b. Tissue profile of C/EBPα mRNA expression by qPCR analysis after 7 days of doxycycline chow diet feeding. n = 4 male mice (control group), n = 2 male mice (Adn-C/EBPα−/− group). This experiment is representative of two independent experiments. c. qPCR analysis of C/EBPα mRNA levels in the adipocyte fraction and SVF fraction of sWAT and eWAT (left) and of C/EBPβ mRNA level in the adipocyte fraction of sWAT and eWAT (right) after 7 days of doxycycline chow diet feeding. n = 3 male mice per group. **, P < 0.001 for sWAT adipocyte; *, P = 0.04 for eWAT adipocyte. This experiment is representative of two independent experiments. d. Western-blots of C/EBPα andβ-actin levels in protein extracts of sWAT, eWAT and liver from Adn-C/EBPα−/− mice and their control littermates after 4 days of doxycycline chow diet feeding. These images are representative of two independent western-blots experiments. e. qPCR analysis of C/EBPα, PPARγ, adiponectin and rtTA mRNA levels during day 0–day 6 of SVF differentiation. SVF is extracted from sWAT of Adn-C/EBPαflox/flox mice. These images are from a single experiments. f. Oil Red O staining of SVF differentiation (extracted from sWAT of Adn-C/EBPαflox/flox mice) on days 0–6, as indicated. These images are representative of two independent western-blots experiments. g. CHIP assay in adipocytes differentiated from SVF of Adn-C/EBPαflox/flox mice. Starting from the 8th way of differentiation, doxycycline (10 μg ml−1) was added to the cell growth medium for 4 days to generate C/EBPα free adipocytes and medium was switched to doxycycline free for another two days of before sample collection. CHIP was performed with C/EBPα (left), C/EBPβ (middle) and PPARγ (right) antibodies and the enrichments of Cd36 and C/ebpβ promoter in the immunoprecipitated DNA were tested by qPCR analysis, using 18s as a quality control. n = 310 cm dishes per group for C/EBPα CHIP; n = 310 cm dishes per group for PPARγ CHIP, Cd36; n = 210 cm dishes (control group), n = 310 cm dishes (Adn-C/EBPα−/− group) for PPARγ CHIP, C/ebpβ. n = 210 cm dishes per group for C/EBPβ CHIP. *, P = 0.02 compared to control group. All data represent the mean or mean ± s.e.m. Student’s t-test. This data is from a single experiment.

Supplementary Figure 2 C/EBPα expression is not required for late embryonic adipogenesis of sWAT and BAT.

a,b. Immunofluorescence stains for C/EBPα (green), DAPI (blue) and perilipin (red) in adipocyte nucleus in eWAT (a) or sWAT (b) of 7 weeks old Adn-C/EBPα−/−(E11−P16) male mice and their control male littermates. These images are representative of two independent experiments. c. Whole tissue pictures of pWAT and sWAT from Adn-C/EBPα−/−(E11−P16) female mice and their control female littermates at 7 weeks of age. These images are from a single experiment. d. H&E staining of sWAT and pWAT in Adn-C/EBPα−/−(E11−P16) female mice and their control female littermates. These images are from a single experiment. e. Perilipin (red) and DAPI (blue) immunofluorescence staining of sWAT and pWAT in Adn-C/EBPα−/−(E11−P16) female mice and their control female littermates. These images are from a single experiment. f. Western-blots of C/EBPα andβ-actin levels in protein extracts of BAT from Adn-C/EBPα−/−(E11−P16) male or female mice and their control littermates at 7 weeks of age. These images are from a single Western-blot experiment. g. Representative H&E stains of BAT in Adn-C/EBPα−/−(E11−P16) male (left) or female mice (right) and their control littermates at 7 weeks of age. These images are from a single experiment. h. Representative perilipin (red) and DAPI (blue) immunofluorescence stains of BAT in Adn-C/EBPα−/−(E11−P16) male (left) or female mice (right) and their control littermates at 7 weeks of age. These images are from a single experiment.

Supplementary Figure 3 C/EBPα is not required for terminal development of eWAT.

a. Experimental design: Adn-C/EBPαflox/flox or control littermates (mice contains only Adn-rtTA and C/EBPαflox/flox) were on doxycycline chow diet during P0–P42. b. sWAT and eWAT tissue mass tissue mass of Adn-C/EBPα−/−(P0−P42) male mice and their control male littermates at 8 weeks of age. n = 4 male mice (control group), n = 3 male mice (Adn-C/EBPα−/−(P0−P42) group). Data represent the mean ± s.e.m. This data is from a single experiment. c. Whole tissue pictures of eWAT and sWAT from Adn-C/EBPα−/−(P0−P42) male mice and their control male littermates. These images are from a single experiment. d. Western-blot of C/EBPα levels in protein extracts of eWAT and sWAT from Adn-C/EBPα−/−(P0−P42) male mice and their control male. These images are from a single Western-blot experiment. e. Experimental design: Adn-C/EBPαflox/flox or control littermates (mice contains only Adn-rtTA and C/EBPαflox/flox) were on doxycycline chow diet during E11–P42. f. sWAT and eWAT tissue mass of Adn-C/EBPα−/−(E11−P42) male mice and their control male littermates at 8 weeks of age. n = 4 male mice per group. Data represent the mean ± s.e.m. This data is from a single experiment. g. Whole tissue pictures of eWAT and sWAT from Adn-C/EBPα−/−(E11−P42) male mice and their control male littermates. These images are from a single experiment.

Supplementary Figure 4 C/EBPα and PPARγ specific transcriptional programs in mature adipocytes.

a. Immediate PPARγ-responsive genes clustered and presented as a heat map, based on the log base 2-transformed expression levels for these genes. Adn-PPARγ−/− mice and their control littermates were on 3 days of doxycycline chow feeding (Fig. 3g). P cut-off: 0.05; fold change cut-off: 2. This data is from a single experiment. b. C/EBPα direct responsive genes clustered and presented as a heat map. Adn-C/EBPα−/− mice and their control littermates were on 3 days of doxycycline chow feeding. P cut-off: 0.05; fold change cut-off: 1.5. This data is from a single experiment. c. Experimental design: 10 weeks old Adn-C/EBPαflox/flox male mice and their control male littermates were kept on doxycycline HFD for 3 days or 1 month to generate Adn-C/EBPα−/− mice. RNA from sWAT of all groups after 3 days of doxycycline chow feeding was extracted for microarray analysis. d,e. C/EBPα direct responsive genes clustered and presented by heat map. Adn-C/EBPα−/− mice and their control littermates were on 3 days of doxycycline HFD feeding (d) or 1 month of doxycycline HFD feeding (e) P cut-off: 0.05; fold change cut-off: 1.5. For each group, RNA from each mouse was pooled into 3 samples for microarray analysis. n = 10 male mice per group, HFD 3 days; n = 9 male mice per group, HFD 1 month. This data is from a single experiment.

Supplementary Figure 5 Mature adipocyte specific inducible deletion of C/EBPα alters whole body lipid metabolism during HFD feeding.

a,b. Lipoprotein profiles determined using pooled serum samples by FPLC fractionation from Adn-C/EBPα−/− mice and their control littermates at the 4th week of doxycycline HFD feeding. Triglyceride (a) and cholesterol (b) concentrations were measured in each indicated fraction corresponding to VLDL, IDL/LDL and HDL, respectively (left). The relative triglyceride content in VLDL fractions (a) and cholesterol content in HDL fractions (b) are also shown using the areas under curve from the FPLC profiles (right). n = 6 male mice per group. This experiment is representative of two independent experiments. c. Triglyceride clearance test (20% intralipid, 10 μl per gram of body weight, single gavage) in Adn-C/EBPα−/− mice and their control littermates. n = 9 male mice (HFD control group), n = 5 male mice (HFD Adn-C/EBPα−/− group). **, P < 0.001, compared to HFD control group. Two-way ANOVA. This data is from a single experiment. d. Triglyceride secretion rate were measured in Adn-C/EBPα−/− mice and their control littermates after tyloxapol administration (through tail-vein injection, 600 mg kg−1 body weight). Blood triglycerides were measured at the time points as indicated (left), and the VLDL secretion rate (right) was derived from the slope of the line using least squares regression. n = 3 male mice (HFD control group), n = 4 male mice (HFD Adn-C/EBPα−/− group). **, P = 0.002, < 0.001 for triglyceride levels at 3 h and 4 h; Two-way ANOVA. *, P = 0.008 for VLDL secretion rate, compared to HFD control group. Student’s t-test. This data is from a single experiment. e–g. Circulating NEFA (e), triglyceride (f) and glucose (g) in Adn-C/EBPα−/− mice and their control littermates at 5th week of doxycycline HFD feeding after β3 agonist treatment (1 mg/kg, body weight). n = 7 male mice (HFD control group), n = 12 male mice (HFD Adn-C/EBPα−/− group). *, P = 0.02 for triglyceride level at 120 min; *, P = 0.04, 0.02 for glucose levels at 30 and 60 min, compared to HFD control group. Two-way ANOVA. This data is from a single experiment. h,i. Hepatic triglyceride (TG) (h) and cholesterol (CHO) (i) levels in Adn-C/EBPα−/− mice and their control littermates after 4 weeks of doxycycline HFD feeding. n = 12 male mice (HFD control group), n = 11 male mice (HFD Adn-C/EBPα−/− group). This data is from a single experiment. All data represent the mean ± s.e.m. j–m. Physical activity (j), oxygen consumption (k), RER (l) and food intake (m) were determined for Adn-C/EBPα−/− mice and their control littermates during the 7th week of doxycycline HFD feeding. n = 6 male mice per group. XA: X beam activities; YA: Y beam activities; CenA: Central beam activities. This data is from a single experiment.

Supplementary Figure 6 Inducible deletion of C/EBPα in mature adipocytes during HFD feeding alters ceramide content and expressions of key transcription factors, adipokines and enzymes in WAT.

a–i. Sphingoid base contents were measured by LC/MS/MS method in liver (a), sWAT (d) and eWAT (g) of Adn-C/EBPα−/− mice and their control littermates after 1 month of doxycycline HFD feeding. Ceramides contents were measured by LC/MS/MS method in liver (b), sWAT (e) and eWAT (h) of Adn-C/EBPα−/− mice and their control littermates after 1 month of doxycycline HFD feeding. Sphingomyelin and Ceramide content was measured by LC/MS/MS method in liver (c), sWAT (f) and eWAT (i) of Adn-C/EBPα−/− mice and their control littermates after 1 month of doxycycline HFD feeding. n = 11 male mice (HFD control group), n = 12 male mice (HFD Adn-C/EBPα−/− group). Data represent the mean ± s.e.m. (a) **, P = 0.004; (b) from left to right: *, P = 0.01, **, P = 0.007, *, P = 0.04; (d) from left to right: *, P = 0.04, **, P < 0.001; (e) from left to right: **, P < 0.001, **, P < 0.001, **, P < 0.001, **, P < 0.001, **, P < 0.001, *, P = 0.03, **, P = 0.006, **, P < 0.001, *, P = 0.01; (f) from left to right: *, P = 0.02, **, P < 0.001; (g) from left to right: **, P < 0.001, *, P = 0.02, **, P < 0.001; (h) from left to right: *, P = 0.04, *, P = 0.02, **, P < 0.001, **, P < 0.001, **, P = 0.002, *, P = 0.03, *, P = 0.02; (i) *, P = 0.01. All compared to HFD control group. Student’s t-test. This data is from a single experiment. j–l. mRNA levels of key transcription factors were measured by qPCR, including PPARγ, SREBP1c, SREBP2, ChREBP, LXRα, LXRβ and PGC1-α (j); adipocyte markers adiponectin (ADPN), resistin, CD36, FATP1, CPT1α and Acox1 (k); lipogenic enzymes FAS, ACC, SCD-1 and Elovl6 (l) in sWAT of Adn-C/EBPα−/− mice and their control littermates after 4 weeks of doxycycline HFD feeding. n = 13 male mice per group. Data represent the mean ± s.e.m. *, P = 0.04 for Elovl6; **, P < 0.001 for PPARγ, LXRα, adiponectin, resistin, CD36 and SCD-1. All compared to HFD control group. Student’s t-test. This data is from a single experiment.

Supplementary Figure 7 Long-term, but not acute C/EBPα deletion on HFD shares common pathways with acute PPARγ deletion on chow diet.

Comparison analysis across the Canonical Pathway Analysis based on the four groups of arrays is shown by heat maps based on z-score. As indicated in Supplementary Fig. 4, the four groups are Adn-PPARγ−/−. mice after 3 days of doxycycline chow treatment; Adn-C/EBPα−/− mice after 3 days of doxycycline chow treatment, 3 days of doxycycline HFD or 1 month of doxycycline HFD. This data is from a single experiment.

Supplementary Figure 8 Mature adipocyte specific inducible C/EBPα knockout alters whole body glucose and lipid metabolism in ob/ob mice.

a. Magnetic resonance imaging analysis from representative ob/ob Adn-C/EBPα−/− mice and their ob/ob control littermates at the 8th week of doxycycline chow diet feeding. These images are from a single Western-blot experiment. b. GTT were performed during the 8th week of doxycycline chow diet feeding on ob/ob Adn-C/EBPα−/− mice and their control littermates, the bar graph represents the area under the curve for the blood glucose levels from glucose tolerance test measurements. n = 5 male mice per group. *, P = 0.02 for GTT 60 min, two-way ANOVA. *, P = 0.03 for GTT AUC compared to ob/ob control group, Student’s t-test. All data represent the mean ± s.e.m. This data is from a single experiment. c. Triglyceride secretion rates were measured on ob/ob Adn-C/EBPα−/− mice and their ob/ob control littermates after tyloxapol administration (through tail-vein injection, 600 mg kg−1 body weight). Blood triglycerides were measured at the time points as indicated (left), and the VLDL secretion rate (right) was derived from the slope of the line using least squares regression. n = 4 male mice (ob/ob control grouP), n = 5 male mice (ob/ob Adn- C/EBPα−/− grouP). **, P = 0.003, < 0.001 for triglycerides levels at 3 h and 4 h, two-way ANOVA; *, P = 0.03 for VLDL secretion rate, compared to ob/ob control group, Student’s t-test. All data represent the mean ± s.e.m. This data is from a single experiment. d–f. Oxygen consumption (d), RER (e) and physical activity (f) were determined for ob/ob Adn-C/EBPα−/− mice and their ob/ob control littermates during the 6th week of doxycycline HFD feeding. n = 6 male mice per group. Data represent the mean ± s.e.m. This data is from a single experiment.

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Wang, Q., Tao, C., Jiang, L. et al. Distinct regulatory mechanisms governing embryonic versus adult adipocyte maturation. Nat Cell Biol 17, 1099–1111 (2015). https://doi.org/10.1038/ncb3217

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