This study demonstrates that Plac8 is required for in vitro white fat adipogenesis. In vivo, however, Plac8 is dispensable for the formation of white adipocytes but its absence leads to obesity with apparently fewer but greatly enlarged adipocytes. The defect in thermoregulation and increased adiposity are, however, not associated with changes in glucose homeostasis nor in plasma free fatty acid and triglyceride levels.
is expressed in both white and brown fat and in both tissues it is enriched in the stromal-vascular fraction containing preadipocytes. General Plac8
inactivation in mice leads to a phenotype of cold intolerance that can be explained by impaired fat oxidation and thermogenesis by brown adipose tissue 
. The late-onset obesity may, however, result from combined defects in both brown and white fat differentiation and function. Here, our studies aimed to determine the role of Plac8
in white adipogenesis and in white fat in Plac8−/−
In vitro, absence of Plac8
expression prevented differentiation into adipocytes of 3T3-L1 preadipocytes, of NIH 3T3 cells, and of MEF derived from Plac8−/−
mice. Kinetics analysis of transcription factor expression upon induction of 3T3-L1 adipocytes differentiation revealed that Plac8
expression peaked at ~3 hours, a kinetics similar to that of Krox20
expression. Analysis of Krox20
, and C/EBPβ
expression in 3T3-L1 cells in which Plac8
was knocked down showed that their expression was reduced. In vivo, Plac8−/−
visceral and subcutaneous fat tissues showed reduced expression of C/EBPβ
. These data therefore suggested that Plac8
is an upstream regulator of the adipogenic transcription cascade. This is in agreement with our studies in BAT preadipocytes, which showed that Plac8
is an upstream regulator of C/EBPβ
. This was also confirmed by demonstrating that Plac8
activated the transcriptional activity of a C/EBPβ
promoter reporter construct expressed in 3T3-L1 adipocytes. Quantitative RT-PCR analysis also showed that absence of Plac8
led to a reduced expression of Klf4
, two factors required early for induction of adipogenesis 
and which acts, at least in part, through induction of C/EBPβ. Thus, Plac8
appears as an upstream regulator of adipogenesis, acting rapidly after induction of differentiation to trigger the adipogenic transcription cascade.
In our present studies differentiation of 3T3-L1 and NIH3T3 cells could be increased by Plac8
transduction, which ensures a stable protein expression for periods of time that are much longer than the transient induction seen upon induction of preadipocyte differentiation. Because Plac8 has been reported to increase the proliferation of certain cell types 
and that adipogenic differentiation requires growth-arrest, the adipocyte differentiation we observed here may underestimate of the role of Plac8 since it can induce two apparently opposed cellular effects. On the other hand, the adipogenic function of Plac8 depends on its transient interaction with C/EBPβ, an event that immediately follows the induction of differentiation, possibly as a result of posttranslational modifications induced by the differentiation cocktail 
. So far, there is no information about Plac8 posttranslational modifications, or its interaction with posttranslational modifications of other proteins. This information is required to fully understand how Plac8 contributes to increased adipogenesis.
Plac8 is a relatively small protein consisting of 124 amino acids containing an evolutionarily-conserved cysteine-rich domain that directs interaction with other proteins. Because Plac8 does not have any structural motif usually associated with transcription factors and due to its small size, its function in adipogenesis is probably to regulate the cellular localization, function, or stability of other proteins, such as through its binding to C/EBPβ, which is required for the C/EBPβ-Plac8 complex to bind to the C/EBPβ
promoter to induce this gene transcription 
In vivo, Plac8
is not absolutely required for either brown or white fat differentiation since both tissues are present in the Plac8−/−
mice. This observation is analogous to that found, for instance with C/EBPβ
. Suppressed activity of either gene in 3T3-L1 fibroblasts prevents their adipogenic differentiation but knockout of either gene in mice does not prevent appearance of white fat depots 
. In Plac8−/−
mice absence of Plac8
leads to a decreased expression of important adipogenic genes such as C/EBPβ
but not of other genes such as C/EBPδ
. This, however, does not prevent augmented fat storage in this tissue. Obesity is usually associated with increase in both adipose cell number and cell size 
. Interestingly, in Plac8−/−
mice obesity is not associated with increased cell number but our data rather suggest a decrease in total adipocyte number. This suggests, that even though adipogenesis during development may proceed normally, the recruitment of new adipocytes from preadipocytes during the adult age may be defective even though the fat storage capacity of existing adipocytes is preserved. This fat cell enlargement occurs in the absence of changes in lipolytic gene expression and with an opposite regulation of lipogenic gene expression, which were increased in VAT (fas
) whereas these genes and acc-1
were decreased in SCAT. The meaning of this opposite regulation is not yet clear.
Decreased BAT activity and increased white fat mass are thought to favor deregulation of glucose homeostasis. However, in the normal chow fed, 29 weeks-old control, Plac+/− Plac8−/− mice studied here fed and fasted glycemia as well as plasma free fatty acids and triglycerides were indistinguishable. This, therefore, suggest that in the conditions studied the defects in thermoregulation and increased white fat mass were not causing major deregulation of glucose homeostasis. We cannot, however, exclude the presence of subtle deregulations in glucose or lipid metabolism, which would require more detailed analysis.
In summary, our data show that Plac8 is required for in vitro adipogenesis and functions upstream of C/EBPβ and possibly Krox20 and Klf4 in triggering adipogenesis. In vivo, Plac8 is dispensable for the production of WAT and BAT. However, because there are fewer white adipocytes in Plac8−/− mice, this suggests that the recruitment of new adipocytes during adult life may be impaired and that the phenotype of the Plac8−/− mice depends on complex interactions between defects in both white and brown adipose tissues.