The study of the conversion of preadipocytes into mature and functional adipocytes is a rather interesting field and the mechanisms that drive the differentiation of the preadipocytes have received a lot of attention due to an increase in obesity and obesity-related diseases. The mechanisms driving the differentiation are indeed rather complicated, but today it is clear that important transcriptional regulators either repress or stimulate the expression of each other. Important transcriptional regulators of the adipogenic program both in vitro and in vivo include family members of the CCAAT/enhancer-binding proteins (i.e. C/EBP, C/EBP and C/EBP) and the nuclear receptor peroxisome proliferator-activated receptor g (PPAR). Generally, the adipogenic program can be divided into two waves of transcription factors that regulate each other in a sequential manner. Adipogenic stimuli will lead to the initiation of the first wave of transcriptional factors. Some of these factors include C/EBP and C/EBP, Krüppel like factors (KLFs), cAMP response element binding protein (CREB), early growth response 2 (Krox20) and sterol regulatory element-binding protein 1c (SREBP-1c). The transcription factors that are a part of the first wave, can then in turn initiate a second wave by inducing the expression of other transcriptions factors such as C/EBP and PPAR. Transcription factors of the second wave will then induce expression of other adipogenic factors resulting in the mature adipocyte phenotype (Figure 1) [1].
Figure 1. The transcriptional network controlling adipogenesis. Stimulation with a hormonal cocktail containing glucocorticoids, insulin, a cAMP-elevating agent and fetal bovine serum activates the early adipogenic transcriptionfacrtors (green). The first wave of transcription facrtors will then induce a second wave of transcription factors (blue), which then induce the expression of genes required for the adipogenic phenotype. Anti-adipogenic factors (orange) can act on different adipogenic factors and thereby inhibit adipocyte differentiation [1].
The Early Adipogenic Program
In vitro model cells such as 3T3-L1 and 3T3-F442A preadipocyte cell cultures can be induced to differentiate into mature adipocytes by stimulating them with a standard hormonal cocktail containing insulin, a cAMP elevating agent, glucocorticoids such as dexamethasone and growth factors (e.g. from serum in the differentiation media) [1]. Addition of a standard hormonal cocktail leads to inhibition of anti-adipogenic transcription factors such as FOXO1, which makes it unable to inhibit the pro-adipogenic transcription factor PPAR. Furthermore, addition of the standard hormonal cocktail also leads to activation of multiple early pro-adipogenic transcription factors such as CREB, KLF4, Krox20 and glucocorticoid receptor (GR), which then in turn can stimulate the expression of other early adipogenic transcription factors such as C/EBP (Figure 1) [1].
Other transcription factors that may play a crucial role in the early adipogenic gene program is STAT5A, which is activated by a tyrosine phosphorylation induced by growth hormones. It has previously been reported that STAT4A can form a complex with GR in the nucleus in the early phase of adipocyte differentiation and that these two factors together with C/EBP and RXR can bind cooperatively to nearly one thousand transcription factor hotspots, only four hours after induction of differentiation [1].
It has earlier been shown with ChIP-seq (a method used to map transcription factor binding sites at a genome wide scale) that GR can bind transiently and cooperatively with C/EBP to thousands of genomic regions, six hours after inducing cells to differentiate. It has also been demonstrated that these regions get marked transiently by H3K9ac (activating histone marks) in the early adipogenic program that possess enhancer-like features (e.g. binding of p300 co-activators and mediator subunit, Med1) and function as enhancers in reporter assays. All this indicates, that these regions in fact are transiently active enhancer regions and that binding of GR and C/EBP to these regions may drive the differentiation of adipocytes in response to an external stimulus [1]. Studies have also shown that the chromatin of the identified hotspots has a momentarily open structure, which indicates that these hotspots may especially be active during the early stage of differentiation, where they can regulate the cell-cycle progression and transcription factors of the late adipogenic gene program (eg. PPAR [1].
The Late Adipogenic Program
As demonstrated on figure 1, transcription factors (eg. C/EBP) of the first wave are able to directly activate the expression of transcription factors of the second wave of adipogenic transcription factors. Some of the most important factors of the second wave are PPAR and C/EBP. It has earlier been shown that multiple early transcription factor hotspots are found near the gene encoding PPAR. This indicates that several signaling pathways coordinate the expression of PPAR during adipogenesis [1]. The Nuclear receptor PPAR is a key regulator of adipogenesis both in vitro and in vivo. Furthermore, the PPAR family members, such as PPAR, function as obligate heterodimers with RXR [2]. It has been shown through genome-wide profiling that binding of PPAR is especially enriched nearby most induced adipocyte genes and genes linked to lipid and glucose metabolism. The enrichment of PPAR binding near these genes happens to be the case in both differentiating and mature adipocytes, which suggests that PPAR are not only a regulator of adipogenesis, but also necessary for the achievement of the mature adipocyte phenotype [1].
Besides enrichment of PPAR at PPAR binding sites, C/EBP ChIP-seq and ChIP-chip showed an extensive colocalization (35-60%) of the, C/EBP and PPAR, which indicates that they may cooperate in the induction of the adipocyte gene program. Studies have also shown that C/EBP is expressed in the late adipogenesis, but at a lower level than during early adipogenesis. Furthermore, C/EBP has a similar binding pattern to C/EBP in mature adipocytes. Mature adipocytes depleted of C/EBP and PPAR failed to maintain the high expression of adipocyte genes nearby PPAR binding sites. Consistently with this, shared binding sites are enriched nearby highly induced adipocyte genes, compared to modestly induced genes. This indicated that cooperation between PPARand results in a great induction of nearby target genes. These results indicate a direct crosstalk between PPARand on chromatin. Cells depleted of either PPARor did not affect the binding of the other factor to the shared sites that were tested. This suggest that the two factors bind independently of each other to the chromatin in mature adipocytes. Taken together, the results suggest that PPARand are a part of the second wave of transcription factors and that they contribute to the assembly of co-factor complexes at their shared sites to induce the expression of adipocyte genes [1].
In this study, the expression of different transcription factors has been studied on RNA level through cDNA-qPCR and on protein level through western blotting. These basic experiments were preformed to shine a light on when different transcription factors, such as C/EBP, C/EBP and RXR are expressed during differentiation of 3T3-L1 adipocytes. Other experiments such as ChIP-qPCR was also carried out, which provided an idea of when transcription factors bind to a specific region of DNA. Results will be discussed later in the report.
PPAR Family Member Proteins and their tissue distribution pattern
Some of the most important transcription regulators of different metabolic programs, in response to nutritional inputs and repressors of proinflammatory gene expression, are the PPAR family member proteins. The PPAR family members consist of three different isotypes, PPAR which is encoded by the PPARA gene, PPAR which is encoded by the PPARG gene and PPAR which is encoded by the PPARD gene. These different transcription factors are also distributed differently in tissues and have different ligand specificities. These distinct functions of the three isoforms make it interesting to study the expression of each isotype in different tissues. The function of the isotype PPAR has mainly been characterized in liver tissue, where its main purpose is to regulate the adaptive response to fasting by controlling processes, such as fatty acid transport, -oxidation of fatty acids and ketogenesis, when activated by its ligands (e.g. Unsaturated fatty acids). Moreover, it is also expressed in BAT, kidney and heart tissue. The expression of the isotype PPAR is highest in adipose tissues (e.g. WAT and BAT) and plays an essential role in the acquisition and maintenance of the mature fat-storing and adipokine-secreting adipocyte phenotype. At last, the isotype PPAR is highly active in skeletal muscle, where it can respond to exercise by regulating fatty acid catabolism and the glycolytic-to-oxidative muscle fiber switch. Activation of PPAR can also improve the lipid homeostasis, prevent weight gain and increase insulin sensitivity. PPAR is also present in tissues such as BAT, WAT, kidney and heart tissue [5].
In this study, the expression of the PPAR family proteins in respectively epididymal white adipose tissue (WAT), intrascapular brown adipose tissue (BAT) and in lever tissue from mice, has been studied on RNA level through cDNA-qPCR and on protein level through western blotting. These experiments were preformed to shine a light on the distribution pattern of the PPAR isotypes in the above-mentioned tissues. Results will be discussed later in the report.