One of the most distinctive phenotypes caused by haploinsufficiency of COUP-TFII in mouse was the impairment of WAT development. The importance of the precise control of COUP-TFII expression levels was also evident in in vitro analyses as well. In addition, we demonstrated that reduction of COUP-TFII expression in 3T3L1 cells results in an increase of Wnt signaling, which is known to be a repressive factor for adipogenesis. Furthermore, we used an inducible system to knockout COUP-TFII in mice and demonstrated that COUP-TFII is essential for the proper formation of adipocytes ( bottom), supporting the positive role of COUP-TFII in adipogenesis. Taken together, the in vivo and in vitro observations indicate that COUP-TFII is required for adipocyte differentiation. The important role played by COUP-TFII in adipocyte differentiation is also consistent with the finding that seven-up
, the Drosophila
ortholog of COUP-TFII, is important for fat body development in flies (Hoshizaki et al., 1994
During the preparation of this manuscript, Xu et al. reported that COUP-TFII is critical in adipocyte differentiation from 3T3-L1 (Xu et al., 2008
). Contrary to our data, their data showed that COUP-TFII plays a negative role in adipogenesis. Over-expression of COUP-TFII reduced and under-expression of COUP-TFII induced adipogenesis in 3T3L1 cells. Since they used a retrovirus system to carry out their studies and we did not, we repeated our experiment using the same viral system to knock down COUP-TFII in 3T3-L1 cells, and the result is consistent with our data showing that down regulation of COUP-TFII resulted in decreased adipocyte differentiation (Figure S2
). In any event, our in vitro data from 3T3-L cells either using siRNA knocking down or a retroviral system is consistent with those from COUP-TFII null MEFs as well as those phenotypes observed in vivo in COUP-TFII+/-
mice. The exact reason for the difference is not clear. Since a transient increase in COUP-TFII expression is observed 4 to 24 hours subsequent to induction of differentiation in 3T3-L1 cells (Fu et al., 2005
and the current data), it is likely that COUP-TFII is needed for adipogenesis at an early expansion phase and its presence at late time points during differentiation may be inhibitory. Therefore, the different times when COUP-TFII was knocked down may explain the difference between Xu's results and ours.
In the mouse, the characteristic morphology of adipocytes develops during the first 24 hr after birth, and there is little or no lipid accumulation at the time of birth (Ailhaud et al., 1992
). After rapid development, however, differentiation of adipocytes from preadipocytes is believed to continue throughout the lifetime of an animal (Rosen, 2002
). Adipocytes develop from mesenchymal stem cells that are characterized by multipotency. The high expression level of COUP-TFII in mesenchymal cells indicates that this transcription factor may play a role in lineage determination. The lower WAT mass in COUP-TFII+/−
mice and the reduced adipocyte differentiation seen in COUP-TFII
null MEFs and COUP-TFII knock down 3T3-L1 cells are consistent with COUP-TFII's role in early adipocyte differentiation, although the present experiment cannot distinguish COUP-TFII's role in preadipocytes from an effect on lineage commitment. To further elucidate the role of COUP-TFII in lineage determination and early differentiation, it will be important to examine the activity of COUP-TFII in a model in which multipotent stem cells differentiate into preadipocytes.
mice are lean and exhibit markedly improved glucose tolerance due to increased peripheral tissue insulin sensitivity (, ). Moreover, they are resistant to high-fat diet induced obesity (), and age-related weight gain (Figure S4A
). Here we show that COUP-TFII+/−
mice are resistant to the impaired glucose tolerance and poor insulin responsiveness observed in wild-type mice fed a high-fat diet (). Markedly improved glucose tolerance was observed in aged COUP-TFII+/−
mice while as compared with control mice (Figure S4B
). Hyperinsulinemic-euglycemic clamps revealed that increased whole-body insulin sensitivity in COUP-TFII+/−
mice resulted from both improved skeletal muscle and white adipose tissue glucose disposal (). Taken together, this data indicates that the increased glucose tolerance observed in COUP-TFII+/−
mice is due to increased insulin responsiveness. Thus, COUP-TFII may be an important modulator of potential linkages between adipocyte metabolism and whole-body glucose tolerance.
Since the total body fat mass in COUP-TFII+/−
mice on a standard diet is reduced by 30% in comparison with wild-type mice () without any significant change in food consumption (), a net increase in energy expenditure must exsist. Furthermore, COUP-TFII+/−
mice appear to be able to up-regulate energy dissipation when fed with a high-fat diet, as demonstrated by the reduced weight gain (), as well as a relative resistance to diet-induced insulin resistance (). One possible mechanism by which this could be achieved is through the upregulation of mitochondrial biogenesis, which could provide a means for an increased flow of FFA to the mitochondria. An increase in density and/or biological activity of mitochondria, as reflected by the increased levels of COXIV and PGC-1α (), would further enhance this process. The relevance of this hypothesis is underscored by the fact that COUP-TFII+/−
mice exhibit increased mitochondrial volume density in their WAT ( and Figure S6
) in association with their increased oxygen consumption (). Our data shows that the proteins of the mitochondrial electron transport chain are negatively controlled by COUP-TFII, and the appearance of the uncoupling protein UCP1 was observed in WAT of COUP-TFII+/−
mice (). UCP1 is normally expressed only in brown fat that is believed to divert the energy derived from mitochondrial electron transport that would normally generate ATP into heat production. This uncoupling effect greatly accelerates oxygen consumption. In the white adipose tissue, COUP-TFII might act normally to attenuate the expression of UCP1 and genes involved in fatty acid oxidation and mitochondrial respiration.
Taken together, phenotypic analysis using the COUP-TFII haploinsufficiency mouse model indicates an important role of this gene in WAT development and metabolism. Although use of this model might be limited for overall phenotypic analysis, it could reveal the global impact of a gene dosage effect on adipogenesis. Detailed molecular and cellular mechanisms could be elucidated by conditional gene targeting approaches, using a specific Cre recombinase to delete COUP-TFII at different stages during adipocyte differentiation.
In summary, the present study discovered an important role played by COUP-TFII in adipocyte differentiation in vivo. Moreover, COUP-TFII+/- mice display enhanced glucose tolerance and insulin sensitivity compared with control mice; data presented here demonstrates that COUP-TFII likely plays a key role as a metabolic regulator. Then, the pharmacological manipulation of COUP-TFII may allow individuals to lose weight while maintaining a normal caloric intake and improved insulin sensitivity. In this way, COUP-TFII is a potential drug target candidate for the treatment of obesity, insulin resistance, and type 2 diabetes.