The ability to differentiate was maintained through multiple passages longer in facial preadipocytes than abdominal preadipocytes (Fig. 14.2). It was determined that facial preadipocytes may be induced to differentiate at a passage greater than 10. The morphological and molecular characteristics at passage 11 were similar to those after the third passage (Fig. 14.3a). In Fig. 14.3b, quantitative polymerase chain reaction (qPCR) analysis demonstrated that the expression of the major adipocyte markers: fatty acid binding protein 4 (FABP4), PPAR-γ, and fatty acid synthase (FAS; glucose transporter 4 (GLUT4), G-protein coupled receptor 81 (GPR81), leptin, and adiponectin as well but not shown) are similarly and significantly induced during differentiation of both facial and abdominal preadipocytes. The late-passage (passage 11) facial preadipocytes did not have significantly different expression levels of the adipogenic or the lipolytic genes tested (more than 20 genes), and these expression levels were similar to those of differentiated early-passage (passage 3–5) abdominal cells.

On day 14 or 15 after adding the differentiation media, preadipocyte cultures became fully differentiated adipocytes, as determined by morphology and adipocyte marker gene expression (FABP4, GLUT4, and PPAR-γ). To test the functional response of these cells, passage 3–5 facial and abdominal preadipocytes were treated with 2 µM of isoproterenol, a β₂-adrenergic receptor agonist (Fig. 14.4). After 5 h, lipolytic rate measured by glycerol release was twofold greater in abdominal than facial adipocytes (Fig. 14.4a). After 1 week of exposure to isoproterenol, facial preadipocytes retained more lipid droplets than abdominally derived cells (Fig. 14.4b). Histological examination revealed more lipid droplets of greater diameter in the facial cells than in the abdominal adipocytes.

An array of genes associated with lipolysis was then tested to examine the possible differences in expression levels between facial and abdominal cells (Fig. 14.4c). Among the 25 genes tested, the expression of the β₂-adrenergic receptor (ADRB2) was significantly different between facial and the abdominal adipocytes. The lower expression levels of ADRB2 from facial adipocytes compared with abdominal adipocytes was consistent with our finding of reduced lipolytic rate in facial fat cells (Fig. 14.4a), suggesting the underlying mechanism.

The differences noted above in expression of the β₂-adrenergic receptors in differentiated preadipocytes from abdominal and facial sources were identified using cultured adipocytes. Further the differences in gene expression observed between these tissue sites also existed in vivo. The gene profiles using tissue obtained from age-matched individuals (45–60 years old) undergoing either facelift or abdominoplasty demonstrated that most gene markers were expressed at similar levels in tissues from both sites. As shown in Fig. 14.5a, significantly less expression of FAS, CPT2, and GLUT4 in facial versus abdominal adipose tissue was observed. Although β₂-adrenergic receptor expression levels was lower in facial versus abdominal adipose tissue, the difference was not statistically significant. The expression the β₃-adrenergic receptor was detected in abdominal adipose tissue, but was completely undetectable in the facial adipose tissue (Fig. 14.5b).

Microarray analysis identified unknown genes that were regulated depot specifically; in five human abdominal adipocytes and four facial. Depot specific patterns of gene expression were observed by principle component analysis (PCA), abdominal samples were separated by facial samples in general, based on the overall expression profiles (Fig. 14.6a). Total of 283 probes differentially regulated between the two depots were identified (Fig. 14.6b). Around 126 probe sets were significantly upregulated in abdominal fat as compared to facial fat, with fold change not less than 2 and the overall FDR less than 0.05. Using the same criteria, 157 probe sets were significantly downregulated in abdominal fat as compared to facial fat (Fig. 14.6b). The detail expression profiles of these 283 probe sets are also illustrated in this heat map Fig. 14.6b. The lists of the top ten genes regulated differentially were summarized in Table 14.1. Interestingly, the majority of upregulated genes in abdominal compared to facial adipocytes, in other words, significantly downregulated in facial were the HOX transcription factors, which have also been identified in other depot comparisons; for example, abdominal versus gluteal regions (Gehrke et al. 2013).