Adipose depots possess unique developmental gene signatures

Abstract

We have previously demonstrated that subcutaneous and intra-abdominal adipose tissue show different patterns of expression for developmental genes (Shox2, En1, Tbx15 Hoxa5, Hoxc8, and Hoxc9), and that the expression level of Tbx15 and Hoxa5 in humans correlated with the level of obesity and fat distribution. To further explore the role of these developmental genes in adipose tissue, we have characterized their expression in different adipose depots in mice, and studied their regulation in obesity and by fasting. Developmental and adipogenic gene expression was compared in two subcutaneous and three intra-abdominal white adipose tissue (WAT) depots as well as brown adipose tissue (BAT) from lean or obese mice in a fed or fasting state. Each of these six adipose depots display a unique pattern of developmental gene expression, whereas expression of adipogenic transcription factors PPARgamma2 C/EBPalpha, beta, and Delta showed constant expression levels in all depots. Expression levels of developmental genes were similar in obese (ob/ob and high-fat diet (HFD)) and lean mice in most depots. Fasting systematically decreased expression of Hoxc8, PPARgamma2, and increased C/EBPDelta in both lean and ob/ob mice, but produced only variable changes in the expression of other developmental and adipogenic genes. These data indicate that each fat depot has a unique developmental gene expression signature, which is largely independent of nutritional state. This finding further supports a fundamental role of developmental genes in fat distribution and the development and/or function of specific adipose tissue depots.

Figure 1

Expression pattern of developmental genes and adipogenic transcription factors in the different fat depots of C57BL/6 mice. (a) White adipose tissues from two subcutaneous fat depots—one from the flank (Flk) and the other from the interscapular area of the back surrounding the brown fat pad (Bck), three intra-abdominal fat depots from epididymal fat (Epd), perirenal fat (Prr) and mesenteric fat (Msn) and interscapular brown adipose tissue (BAT) were collected from 14-week-old male C57BL/6 mice under random feeding condition. Total RNA was extracted, and cDNA was synthesized. Gene expression of developmental genes Hoxa5, Hoxc8, Hoxc9, Shox2, En1, and Tbx15 was quantified by real-time PCR using the standard curve method, with expression in flank depot assigned a value of 100 arbitrary units. Five independent samples each from a different mouse (n = 5) were each analyzed in duplicate or triplicate. Differences between all depots were statistically analyzed by one-way analysis of variance with the Games-Howell post hoc test. The bar indicates significant difference (P < 0.05) between depots. Gene expression levels in two subcutaneous fat (Flk, Bck) and BAT are shown by the open bars, and the gene expressions in three intra-abdominal fat depots (Epd, Prr, Msn) are shown by the filled bars. Each fat depot shows a unique pattern of distribution. (b) Gene expression of four adipogenic transcription factors of Cebpb, Cebpd, Cebpa, and Pparg2 were assessed by quantitative real-time PCR using the same methods and samples indicated above.

Figure 2

Comparison of expression of the developmental genes and adipogenic transcription factors in the different fat depots of C57BL/6 and ob/ob mice. (a) Total RNA was extracted from the six different fat depots in 10-week-old male ob/ob mice (on a C57BL/6 background) under random feeding conditions, and gene expressions of Hoxa5, Hoxc8, Hoxc9, Shox2, En1, and Tbx15 assessed as described in the legend of Figure 1 and compared to that in lean C57BL/6 mice. Note that the unique distribution of the developmental transcription factors observed in lean mice was conserved in ob/ob mice, with the exception of Hoxc8. (b) Gene expressions of the adipogenic transcription factors, Cebpb, Cebpd, Cebpa, and Pparg2 in ob/ob vs. lean mice. Gene expression in adipose tissue of C57BL/6 mice (open columns) and ob/ob mice (filled columns) was assessed as described in Figure 1b. Differences in gene expression in the same depot between C57BL/6 mice and ob/ob mice from five independent samples each from a different mouse (n = 5) were analyzed by Student's t-test. *statistical significance at P < 0.05.

Figure 3

Comparison of expression of the developmental genes the different fat depots of C57BL/6 mice in LFD vs. HFD. Three-week old C57Bl6 male mice were fed for 7 weeks with a normal chow diet (LFD, opened columns) or with a high-fat diet (HFD, filled columns). Gene expression of Shox2, Tbx15, En1, Hoxc9, Hoxc8, and Hoxa5 was measured in the six different fat depots of both LFD and HFD mice by quantitative RT-PCR. Note that the unique distribution of the developmental transcription factors observed in lean LFD mice was conserved in obese HFD mice. Differences in gene expression before and after fasting in the same depot from five independent samples each from a different mouse (n = 5) were analyzed by Student's t-test (*significance at P < 0.05).

Figure 4

Effect of fasting on gene expression of developmental and adipogenic transcription factors genes in C57BL/6 mice and ob/ob Mice. Expression of the six developmental transcription factors Shox2, En1, Tbx15, Hoxc9, Hoxc8 Hoxa5 (a,b) and four adipogenic transcription factors Cebpb, Cebpd, Cebpa, and Pparg2 (c,d), were measured in six different fat depots before and after fasting in both lean C57BL/6 mice (a,c) and ob/ob mice (b,d). Differences in gene expression before and after fasting in the same depot were analyzed by Student's t-test. Note that Hoxc8 in white fat pads shown decreased trend by fasting in both C57BL/6 mice and ob/ob mice. Expression of each gene in the different fat depots is indicated as open circles (Flk), filled circles (Bck), open squares (Epd), filled squares (Prr), open triangles (Msn), open diamonds (BAT). Differences in gene expression before and after fasting in the same depot from five independent samples each from a different mouse (n = 5) were analyzed by Student's t-test (*significance at P < 0.05).