PITX2 is enriched in highly adipogenic brown preadipocytes

Abstract

Brown adipose tissue (BAT) serves as a key heat-producing organ required to maintain body temperature and homeothermy in mammals. BAT recruitment and activation deeply impacts metabolic homeostasis in both mice and humans. Despite recent advances, the nature of the factors that functionally characterize brown adipose precursors are still incompletely characterized. Here, we provide a comprehensive transcriptomic analysis of brown preadipocyte cell lines with low or high adipogenic potential. Using this resource, we report the identification of paired-like homeodomain 2 (PITX2) as a transcription factor highly enriched in committed brown preadipocytes.

Figure 1. PITX2 is a transcription factor highly expressed in committed brown preadipocytes

(A) Examples of brown preadipocytes showing either low or high adipogenic capacity. Pictures were taken from the same well 5 days following the induction of adipogenic differentiation. Cells were stained with DAPI (blue) to stain the nuclei. (B) Schematic presentation of the experimental procedure used to isolate and study clonal lines of brown preadipocytes. (C) Triglyceride accumulation measured in 26 clonal lines of brown preadipocytes following the induction of differentiation. Lipid content was measured per well (n=3/cell line) 5 days after the induction of differentiation. (D) Oil red O staining of representative Low and High adipogenic lines 5 days following the induction of differentiation. (E) Quantitative RT-PCR analyses of adipogenic genes measured in Low (n=8) and High lines (n=7) 5 days after the induction of adipogenesis. (F) Heatmap presenting the differential expression profile of several genes between Low (n=8) versus High (n=8) adipogenic lines measured by microarray. The clones are presented in ascending order of their adipogenic capacity. This experiment was performed in pre-confluent cells. (G) Quantitative RT-PCR analyses of Pitx2 isoforms expression in Low (n=8) and High (n=8) adipogenic lines at pre-confluence. (H) Western blot analysis of PITX2A protein levels in a subset of Low and High adipogenic clonal lines (n=6/lines). S6K1 was used as a loading control. (I) Quantitative RT-PCR analysis of Pitx2 gene expression in 3T3-L1 preadipocytes, in a Low adipogenic clonal line (4H7), a High adipogenic clonal line (4H10), and C2C12 myoblasts. For all cell lines, a n=4 was used. (J) Western blot analysis of PITX2A protein levels in 3T3-L1 preadipocytes, one Low adipogenic clonal line (4H7), one High adipogenic clonal line (4H10), and C2C12 myoblasts. S6K1 was used as a loading control. (K) Quantitative RT-PCR analyses of Pitx2 , Pitx2a , Pitx2b , Pitx2c , Pparg2 , Ucp1 , and Lpl at sub-confluence (sub) or 0, 1, 3, 5, and 7 days following the induction of adipogenic differentiation (n=4/condition). The parental brown preadipocyte cell line was used in this study. In all panels, data are presented as mean ± SEM. In panels E and G, significance was determined by 2-tailed, unpaired t test. In panels I and K, significance was determined by one-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test. In all panels, significance is represented as follow *P < 0.05, **P<0.01, ***P<0.001, ****P<0.0001.