C/EBP α is Essential for Gonadal but Not Inguinal White Adipose Tissue Formation in Mice

FIGURE 1. Cebpa_ASKO mice have altered gWAT development. (A) Schematic of Cebpa_ASKO mouse alleles and adipocyte differentiation knockout timeline. (B) Taqman qPCR for Cebpa in gWAT from male 10‐week‐old Cebpa_fl/fl and Cebpa_ASKO mice (n = 4–5). (C) Body weight in male 10– to 12‐week‐old Cebpa_fl/fl and Cebpa_ASKO mice (n = 5–7). (D) Macroscopic image of adipose tissue depots from male 10‐week‐old Cebpa_fl/fl and Cebpa_ASKO mice. (E) Macroscopic image of gWAT attached to gonads from male 10‐week‐old Cebpa_fl/fl and Cebpa_ASKO mice. (F) Mass of gWAT from male 10– to 12‐week‐old Cebpa_fl/fl and Cebpa_ASKO mice (n = 5–7). (G) H&E stain of developing gWAT appendage from Cebpa_fl/fl and Cebpa_ASKO neonates. (H) Identification and quantification of adipocytes by Adiposoft in one section of developing gWAT appendages. All mice were chow‐fed. Student's t‐test was used to analyze results (*p < 0.05, *****p < 0.0001). Schematic created with Biorender. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2. Cebpa_ASKO iWAT develops despite Cebpa loss. (A) Taqman qPCR for Cebpa in iWAT from Cebpa_fl/fl and Cebpa_ASKO mice (n = 5–7). (B) Western blot for C/EBPα on iWAT homogenates from 10‐week‐old male mice. (C) Mass of iWAT from 10‐ to 12‐week‐old male Cebpa_fl/fl and Cebpa_ASKO mice. (D) Representative H&E stain of iWAT from 10‐week‐old male mice. (E, F) Quantification of cell size by Adiposoft and an estimate of adipocyte number by the Goldrick formula (n = 3–4). (G) Taqman qPCR for Cebpa during differentiation in iWAT SVF. (H) Neutral lipid droplets during SVF‐derived adipocyte differentiation at D0, D2, D5, D7, and D9 were visualized by Oil Red O staining. (I) Quantification of extracted stained lipids from panel H (n = 2). (J) Leptin measured via ELISA in plasma of 4‐h fasted Cebpa_ASKO mice (n = 5–7). Student's t‐test was used to analyze results (*p < 0.05, **p < 0.01, ****p < 0.0001). [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3. Cebpa_ASKO mice do not expand adipose on a high‐fat diet (HFD). (A) Growth curve of Cebpa_fl/fl and Cebpa_ASKO male mice fed HFD starting at 10–12 weeks of age. (B) Body weight of Cebpa_fl/fl and Cebpa_ASKO mice after 17 weeks of HFD feeding. (C) Weight gain in Cebpa_fl/fl and Cebpa_ASKO mice after 17 weeks of HFD feeding measured as percent change in body weight. (D) MRI measured fat mass and lean mass in a separate cohort of Cebpa_fl/fl and Cebpa_ASKO mice after 15 weeks of HFD feeding. (E) Macroscopic image of adipose depots of Cebpa_fl/fl and Cebpa_ASKO mice after 20 weeks of HFD feeding. (F–H) Masses of gWAT, iWAT, and other adipose depots from panel E reported by percent body weight (n = 6–7). Student's t‐test was used to analyze results (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4. Cebpa_ASKO iWAT is dysfunctional. (A) Number of differentially expressed genes in bulk RNA‐seq of control and Cebpa_ASKO iWAT total tissue RNA. (B) Volcano plot of DESeq2 analysis of bulk RNA‐seq data from Cebpa_fl/fl and Cebpa_ASKO iWAT (n = 3). (C) Gene set enrichment analysis for GO terms from bulk iWAT RNA‐seq data. (D) Taqman qPCR for Adipoq in iWAT from Cebpa_fl/fl and Cebpa_ASKO mice (n = 5–7). (E) Adiponectin measured via ELISA in plasma of 4‐h fasted Cebpa_ASKO mice (n = 5–7). (F) Adiponectin (Acrp30) protein levels measured via Western blot in lysates from Cebpa_fl/fl and Cebpa_ASKO iWAT. (G) Identification of upstream regulators most likely to contribute to differences in gene expression using Ingenuity Pathway Analysis. (H) Taqman qPCR for Pparg in whole iWAT (n = 5–6). (I) Western blot analysis of PPARγ in homogenates of whole iWAT (n = 4). (J) Densitometry analysis of panel I, normalized to vinculin. (K) Nonesterified fatty acid concentration measured in media from iWAT SVF‐derived adipocytes treated with vehicle or CL‐316,243, normalized to triglyceride (TG) mass of each sample (n = 2). (L) Western blot analysis of lipolysis markers in homogenates of SVF‐derived…

FIGURE 5. Cebpa_ASKO mice have whitened BAT. (A) Taqman qPCR for Cebpa in RNA extracted from whole BAT (n = 5–7). (B) BAT mass reported as percentage of body weight in Cebpa_fl/fl and Cebpa_ASKO mice. (C) Representative H&E stain of BAT from Cebpa_fl/fl and Cebpa_ASKO mice. (D) Taqman qPCR for Cidea, Lep, Ppargc1a, Prdm16, and Ucp1 expression in RNA extracted from whole BAT (n = 5–7). (E) Representative H&E stain of BAT from Cebpa_fl/fl and Cebpa_ASKO mice fed HFD for 20 weeks. (F) Macroscopic image of BAT from panel E. (G) BAT mass reported as percentage of body weight in Cebpa_fl/fl and Cebpa_ASKO mice after 20 weeks of HFD feeding. Male mice were 10–12 weeks old and chow‐fed unless otherwise noted. Student's t‐test was used to analyze results (*p < 0.05, ***p < 0.001, ****p < 0.0001). [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 6. Cebpa_ASKO mice have increased lipid accumulation in their livers. (A) Liver mass reported as percentage of body weight from Cebpa_fl/fl and Cebpa_ASKO mice. (B) Hepatic triglycerides and (C) cholesterol measured in liver homogenates (n = 5–7). (D) Plasma triglycerides and (E) cholesterol were measured in 4‐h fasted mice (n = 5–7). (F) Cholesterol FPLC profile of pooled plasma (n = 5–6) from 4‐h fasted male Cebpa_fl/fl and Cebpa_ASKO mice. (G) Pie chart of the number of differentially expressed genes in bulk RNA‐seq. (H) Volcano plot of DESeq2 analysis of bulk RNA‐seq data from Cebpa_fl/fl and Cebpa_ASKO livers (n = 5). (I) Gene set enrichment analysis for GO terms from RNA‐seq data. (J) Heat map of expression of genes clustered in the fatty acid metabolic process GO term from bulk RNA‐seq. All mice were 10–12 weeks old and chow‐fed. Student's t‐test was used to analyze results (*p < 0.05, **p < 0.01). [Color figure can be viewed at wileyonlinelibrary.com]