Adipocyte differentiation

Cytoskeleton, tight junction and cell adhesion reorganization are well known features of adipogenesis (224). The expression profile of different markers of adipocyte differentiation such as aP2, CD36 and SREBP1c, showed that PPARγ +/- adipocytes are less fully differentiated than their wild type counterparts (289).

During adipogenesis, extracellular matrix (ECM) remodelling defines the onset of the differentiation process, characterized by the conversionfrom the fibronectin-rich stromal matrix

RESULTS-PPARγγγγ and adipose tissue integrity

Neuroepithelial Cell Transforming Gene 1 SH3 Ankyrin Domain Gene 3

G protein Coupled Receptor 56 Melanoma Cell Adhesion Molecule SPARC Related Modular Calcium Binding 1

Transmembrane 4 Superfamily Member 7 Integrin Cytoplasmic Domain Associated Protein 1

Prominin 2

Multiple PDZ Domain Protein Claudin 5

Calcium Binding Protein P 22

Nucleobindin 2 Reticulocalbin Calnexin Calmodulin 2

Shingosine 1 Phosphate Phosphatase 1 Makorin 1

Transformation Related Protein 63

Growth Arrest and DNA Damage inducible 45 gamma

Regulated in Development and DNA Damage Responses 1

Glutathione S Transferase mu 6

Microsomal Glutathione S Transferase 3 Glutathione Peroxidase 1

GenBank Gene Name Symbol R^*

A Desintegrin Metalloproteinase Domain 7 Defensin β 2

Receptor (Calcitonin) Activity Modifying Protein 3

Aldo Keto reductase 1A 1 Acid Phosphatase 5

CEA related Cell Adhesion Molecule 10 Lactotransferrin

Table 3: Genes affected in WAT by the deletion of one PPARγγγγ allele. Their expression reflects deregulation of adipocyte differentiation, cell stress and detoxification and finally of the cell inflammatory and innate immunity reactions, (R*: fold change in heterozygous animals compared to wt mice, whose value is set to 1; threshold 1.3; validated result: reproducibility in 7 out of 9 comparisons). n = 3.

of the preadipocyteto the basement membrane of an adipocyte. The expression of ECM components is highly regulated duringthis process: types I and III collagen, fibronectin, and ß1-integrins are down-regulated, whereas type IV collagen and actin are up-regulated (304,305). During ECM remodelling, adipogenic cells release their cell-ECM adhesion and change their morphology and volume (306). The morphologicaland cytoskeletal reorganizations in the preadipocyte is required for the induction of the expression of lipogenicgenes (307-309). Our microarray analysis showed that the expression of Ank 3, which is a component of the actin cytoskeleton known as being down-regulated during adipogenesis (224), was increased in the PPARγ +/- WAT by 2.6 folds (Table 3).

Besides Ank 3, the expression Pfn 2 which maintains actin in a non adipogenic permissive

RESULTS-PPARγ and adipose tissue integrity

monomeric form (310) was increased by 51 folds in the PPARγ +/- WAT. Furthermore, the expression of proteins controlling the actin skeleton dynamics such as Net 1, and Shank 3 were down-regulated by 2 and 1.8 folds respectively, indicating a destabilization of the actin cytoskeleton in the heterozygous WAT (Table 3)(310-313).

As mentioned previously, proper adipocyte differentiation requires loss of the cellular adhesion (307). Our microarray analysis revealed a deregulation of genes involved in this function, (Table 3). Gpr 56 which is an important member of the adhesion family of G protein coupled receptors was up-regulated by 1.7 folds, whereas Icap 1 which is involved in the integrin dependent signals was down-regulated by 1.5 folds in the heterozygous WAT (314,315). The expression of Icap 1 was also confirmed by qRT-PCR analysis on additional RNA samples, (Fig 7). Other genes involved in these function are deregulated in PPARγ +/- WAT. However, their role in adipocyte differentiation is not completely understood. The deregulation of genes involved in the actin skeleton dynamics as well as in the cellular adhesion suggests retardation in the adipocyte differentiation process.

Besides the morphological and cytoskeleton reorganizations, adipocyte differentiation requires changes in the plasma membrane composition such as a high increase in the caveolae composition and a reduction in the gap junctions (316-319). Claudin 6, which is a member of the claudin family involved in the tight junction, was shown to be required for adipocyte differentiation (317). Our microarray and qRT-PCR analysis showed a 1.7 folds down-regulation of another member of this family called Cldn 5, (Table 3, Fig 7). These results are in agreement with a retardation of the adipocyte differentiation in the PPARγ +/- WAT.

Modulation of the calcium signaling is another feature of the adipocyte differentiation (320).

The intracellular calcium concentration affects adipogenesis. In fact, high concentrations inhibit the early stages of adipocyte differentiation, while promoting both the late stage of differentiation and lipogenesis (321). Several calcium binding proteins such as Nucb 2, Rcn 1, calnexin and calmodulin 2 were up-regulated in the heterozygous WAT by 3.4, 2.3, 1.7 and 1.4 folds respectively. However, their involvement in adipocyte differentiation is largely unknown. Calmodulin 2 might be involved in the inhibition of adipocyte differentiation trough activation of the calcium/calcineurin dependent pathway. Nerveless however, the precise mechanism remains obscure (322).

Figure 7. Expression levels of different genes involved in A.

adipocyte differentiation, B. inflammatory and C.

glucocorticoid signaling pathways in the white adipose tissue.

Integrin Cytoplasmic Domain Associated Protein 1 (ICAP 1), Claudin 5 (Cldn 5); Interleukin 6 (Il-6), Monocyte Chemotactic Protein 1(MCP1), Tumour Necrosis Factor α (TNF-α); 11-β-Hydroxysteroid Dehydrogenase type 1 (Hsd11β1), Glucocorticoid receptor (GR), FK506 Binding Protein 5 (FKBP51) n = 4 * p-value ≤ 0.06

RESULTS-PPARγγγγ and adipose tissue integrity

Adipocyte differentiation requires a growth arrest (224,323). Several genes involved in cell cycle control were deregulated in the PPARγ +/- WAT. However, their roles in adipogenesis have not been studied yet (Table 3). High expression of Spp 1, Mkrn 1 and p65 was shown to induce apoptosis in different cellular models (324-326) and were up-regulated in the heterozygous WAT by 3.1, 2.6 and 2.5 folds, respectively. The tumour suppressor Prss 1 and the regulators of the cell cycle Meox 2 and Ccnd 2 were down-regulated in the heterozygous WAT by 1.7 and 1.9 folds respectively. Ccdn2 expression was shown to be increase during adipogenesis (224). These results show, that there is a deregulation of the cell cycle in the heterozygous WAT. However, further experiments are required to better understand this deregulation.

Our histological analysis using hematoxylin and eosin staining showed, in agreement with published data (245), a 20% decrease of adipocytes size in PPARγ +/- mice WAT (Fig 8 C). These results are in agreement with retardation in adipocyte development.

Our microarray and qRT-PCR analysis revealed important deregulations in the expression of genes involved in several aspects of adipocyte differentiation. These results are in agreement with a retardation of the adipogenic process. However, a better validation of this hypothesis requires a deeper characterization of the differentiation process. Since ECM remodelling during adipogenesis requires up-regulation of actin and collagen I and III expression as well as rearrangement of the actin skeleton from a monomeric to a polymerized form, we propose the characterization of the adipocyte cytoskeleton by immunofluoresence using antibodies against proteins of the ECM such as the actin and collagen I and III.