4. Discussion
4.1 Phenotype of the Fra1 transgenic and the Fra1 knockout mouse
AP-1 acts as a sensor for extracellular signals regulating a variety of cellular processes.
Several mouse models have been generated to investigate the effects of overexpression or deletion of the single AP-1 family members. In this study, we analyzed the function of Fra1 in adipogenesis. We therefore used the Fra1 transgenic mice for gain of function as well as the Ap2-cre Fra1f/f mice for loss of function. The Fra1 transgenic mice express the murine Fra1 gene under the control of the H2-Kb-promoter, leading to expression of the transgene in most tissues. Young Fra1 transgenic mice do not have any overt phenotype. However, starting at the age of 1 month, a reduced growth rate was described (Jochum et al., 2000). Mice overexpressing Fra1 develop an osteosclerotic phenotype caused by an enhanced bone formation as a consequence of an increased number of mature osteoblasts, a phenotype similar to the osteosclerosis described for ∆FosB transgenic mice (Sabatakos et al., 2000). In vitro experiments using primary osteoblasts derived from Fra1tg mice showed that the increased number was not caused by an increased proliferation rate, but due to a cell autonomous accelerated osteoblast differentiation. As a result of the increased bone formation, splenomegaly due to extramedullary hematopoiesis was observed. In addition, Fra1tg mice can develop lung tumors and liver cirrhosis (Jochum et al., 2000; Kireva et al., 2011).
Before analyzing mice overexpressing Fra1, we tried to determine the effect of Fra1 deficiency in adipogenesis. As Fra1 knockout mice are embryonic lethal due to defects in the placenta (Schreiber et al., 2000), Fra1f/f mice have been generated and crossed with MORE-cre mice, leading to a deletion of Fra1 in the entire embryo except in the extraembryonal tissue. MORE-cre Fra1f/f mice were described to be osteopenic due to a decrease in bone matrix formation with a reduced level of osteocalcin, collagen1a2 and matrix Gla protein. No other overt alterations were found in the MORE-cre Fra1f/f mice (Eferl et al., 2004). For our studies, we crossed the Fra1f/f mice with the Ap2-cre mice to obtain a specific deletion of Fra1 in mature adipocytes. We could not observe a clear adipocyte phenotype in Ap2-cre Fra1f/f mice. However, this could be due to the inefficient cre-mediated recombination in the adipose tissue. Thus, we investigated the effect of gain-of-function of Fra1 by analyzing Fra1 transgenic mice.
4.2 Fra1 overexpression decreases adipose tissue mass We found that Fra1 transgenic mice show an age-dependant loss of body weight due to a reduced amount of adipose tissue mass. While the weight of the visceral fat pad is similar in young Fra1 transgenic mice compared to wild-type littermates, the transgenic mice completely loose their visceral fat tissue when aging. To investigate if the cause of the observed lipodystrophy is based on a systemic defect, we analyzed the glucose and lipid metabolism of the Fra1 transgenic mice and compared our results to the lipodystrophic phenotype characterized for the ∆FosB transgenic mice. Indeed, ENO2-∆FosB mice, expressing ∆FosB under the neuron-specific enolase promoter, show a decrease in adipose mass (Sabatakos et al., 2000). However, no effect on adipose tissue mass could be observed by adipocyte- or osteoblast-targeted expression of ∆FosB, indicating that the fat phenotype is not cell-autonomous or caused by increased bone mass, respectively (Kveiborg et al., 2004;
Rowe et al., 2009). Analysis of endocrine parameters of ENO2-∆FosB mice revealed no change in triglyceride levels, but reduced levels of insulin, free fatty acids and fed and fasting blood glucose levels. Additionally, increased glucose tolerance and enhanced insulin sensitivity were observed. Thus, the decrease in white adipose tissue observed in
ENO2-∆FosB mice is most likely caused by systemic metabolic alterations due to an increase in energy expenditure associated with an elevated fatty-acid oxidation in the muscle (Rowe et al., 2009).
In contrast, metabolic studies of Fra1 transgenic mice revealed only few marginal alterations.
As expected for a reduced amount of adipose tissue mass, Fra1 transgenic mice show a reduced level of leptin in the serum while the increased osteocalcin serum levels areconsistent with the increased bone mass. The adipocyte-derived leptin acts via the hypothalamus to regulate food intake and energy expenditure (Yadav et al., 2011). However, the reduced serum level of leptin could not alter the food intake of the Fra1tg mice. Furthermore, the effect of leptin deficiency to increase bone formation (Ducy et al., 2000) was shown to not cause the osteosclerotic phenotype of ∆FosB transgenic mice (Kveiborg et al., 2002).
As it was recently reported, that osteocalcin regulates insulin signaling by increasing pancreatic β-cell proliferation (Lee et al., 2007), insulin secretion and insulin sensitivity (Clemens and Karsenty, 2011), we expected that the increased osteocalcin levels would lead to alterations in the insulin signaling of Fra1tg mice. However, we observed normal levels of β-cell proliferation and of circulating insulin in the Fra1 transgenic mice, probably because of the increased Esp levels described to reduced bioavailability of osteocalcin (Ferron et al., 2010b). This observation of normal insulin levels and unchanged number and volume of pancreatic β-cell islets despite an increased osteocalcin level was also described for ∆FosBtg mice (Rowe et al., 2009). Furthermore, fasting blood glucose levels in the Fra1tg mice were decreased, probably because of their increased sensitivity to insulin.
The only major metabolic alterations in Fra1tg mice are increased levels of circulating triglycerides and of non-esterified fatty acids. Further features typically associated with severe forms of lipodystrophy as described in typical mouse models for lipodystrophy like in A-ZIP/F transgenic mice (Moitra et al., 1998) or mice overexpressing a constitutively active form of SREBP-1c (Shimomura et al., 1998) are type 2 diabetes, insulin resistance as well as hepatic steatosis. This phenotype was not observed in Fra1 transgenic mice. It thus appears unlikely that the lipodystrophy of the Fra1 transgenic mice is due to systemic metabolic alterations.
4.3 Fra1 regulates adipocyte differentiation in a cell autonomous manner
In addition to systemic alterations, cell-autonomous defects can cause a reduction in adipose tissue mass. Adipogenic differentiation in vitro can be induced by stimulation with an adipogenic cocktail consisting of insulin, IBMX and dexamethasone. We could show in studies using a cell line overexpressing Fra1 and primary calvarial osteoblasts of Fra1tg mice that increased levels of Fra1 result in a reduced amount of mature adipocytes as shown by Oil
Red O staining and confirmed by the decreased expression of late adipogenic markers as C/ebpα, Pparγ2 and their transcriptional targets Ap2 and Glut4. The finding of a cell-autonomous defect is further supported by the similar results obtained in studies using an estradiol-inducible Fra1 in a cell culture system.
A shift to an increased osteoblastogenesis at the expense of adipogenesis was described in osteoblast differentiation experiments using cells overexpressing Fra2 (Bozec et al., 2010).
However, in cells overexpressing Fra1 as well as in the adipose tissue of Fra1tg mice, markers for lineage commitment (Pref1, Runx2, Osx1, Sox9 and MyoD) are not changed, nor were the markers for early adipocyte differentiation (C/ebpβ and C/ebpδ), supposing that the differentiation to mature adipocytes rather than adipocyte commitment is regulated by Fra1.
Consistent with an adipocyte maturation defect is the observation of a significant decreased adipocyte size in the visceral fat depots that develop in Fra1 transgenic mice indicating an increased number of immature adipocytes. Thus, we could demonstrate that Fra1 cell-autonomously regulates adipocyte maturation.
4.4 Fra1 directly regulates adipocyte differentiation by reducing C/ebpαααα expression While the expression of genes marking early steps of adipocyte development, as well as several factors involved in later differentiation stages are not affected in vivo, the level of C/ebpα, a key determinant of adipogenesis required and sufficient for terminal adipocyte differentiation of 3T3-L1 preadipocytes (Cao et al., 1991; Lin and Lane, 1994; Wu et al., 1998) as well as for the maintenance of the differentiated phenotype (Mandrup and Lane, 1997) is significantly reduced. Adipocytes lacking C/ebpα display a decreased insulin-regulated glucose uptake as C/EBPα regulates the insulin receptor and the glucose transporter Glut4 leading to reduced lipid accumulation. We could not detect any alterations in the insulin response in vitro by analyzing the phosphorylation of ERK and AKT. However, in differentiation experiments, we observed a reduced accumulation of lipid droplets and a decreased expression of Glut4 in the cells overexpressing Fra1. Consistent with a downregulation of C/ebpα are the experiments using the estradiol-inducible Fra1 cell line where activation of Fra1 blocks the early induction of C/ebpα by the adipogenic cocktail. We also found C/ebpα downregulated in other tissues with increased Fra1 expression, namely the liver, the muscle and the bone, further supporting a key role for C/ebpα repression by Fra1.
Another in vivo model for lipoatrophic diabetes is a transgenic mouse expressing the dominant-negative protein, A-ZIP/F under the control of the Fabp4 (Ap2) promoter. A-ZIP/F forms heterodimers with b-ZIP transcription factors via its leucine zipper and therefore inhibits the binding of C/EBP and AP-1 family members to DNA. Inhibition in activity of these transcription factors exclusively in adipose tissue leads to the ablation of white adipose tissue throughout development (Moitra et al., 1998). Studies overexpressing another dominant negative protein (A-C/EBP) in mice that only inhibits the C/EBP binding to promoter sequences leads to a lack of white adipose tissue in young and a reduced amount of WAT in older mice (Chatterjee et al., 2011). Due to the less severe phenotype of A-C/EBP compared to A-ZIP/F mice, it is proposed that both, AP-1 and C/EBP family members are important for growth and differentiation of white adipose tissue.
In agreement, some AP-1 members such as c-Fos, its viral homologue v-Fos as well as ∆FosB where shown to alter adipocyte differentiation cell-autonomously by regulating transcription factors of the C/EBP family. In particular, c-Fos was described to influence the activity of C/EBP family members in vitro as it can directly bind to C/EBPβ (Hsu et al., 1994). We could also show in initial experiments that overexpression of c-Fos in an adipogenic cell line reduced adipocyte differentiation and decreased the expression level of C/ebpα. However, overexpression of Fra2 in this cell line did not result in reduced adipocyte differentiation or in an altered expression of C/ebpα. Although the expression of adipogenic transcription factors in adipose tissue of ENO2-∆FosB mice was not changed (Rowe et al., 2009), the reduced adipogenic differentiation of primary osteoblast cultures overexpressing ∆FosB was reported to be caused by an interaction of ∆FosB with C/EBPβ resulting in the inhibition of transcriptional activation of C/ebpα (Kveiborg et al., 2004). In contrast, the viral homologue of c-Fos, v-Fos that inhibits adipocyte differentiation in vitro results in a decreased level of transcription as well as inhibited activity of C/EBPα while it does not affect the expression or activity of C/EBPβ (Abbott and Holt, 1997). Therefore, the regulatory role of v-Fos is not mediated by binding to C/EBPβ or C/EBPα. Performing co-immunoprecipitations, we were not able to demonstrate an interaction of Fra1 with C/EBPβ. In contrast, we could show by promoter studies and chromatin immunoprecipitation experiments that the reduced C/ebpα expression level in Fra1 transgenic mice is caused by the inhibition of transcription due to the binding of Fra1 to a putative binding site in the C/ebpα promoter.
4.5 Influence of the reduced amount of mature adipocytes on metabolism
Indeed, a decreased level of C/ebpα can explain the lipodystrophic phenotype observed in Fra1 transgenic animals as consistent observations were reported in C/ebpα knockout strains.
Although C/ebpα deficient mice die due to liver failure (Wang et al., 1995), studies using a C/ebpα knockout mouse with a transgenic expression of C/ebpα under the control of the albumin promoter in order to rescue the liver phenotype(Linhart et al., 2001) as well as a poly IC-inducible C/ebpα knockout mouse model where the postnatal deletion can be obtained in various especially C/EBPα-rich tissues (Yang et al., 2005) demonstrated, that a reduced level of C/ebpα can account for a severe reduction in white adipose tissue mass. However, in accordance with the unchanged weight of other tissues, including brown adipose tissue in the Fra1tg mice, C/EBPα was described to be not required for the postnatal development of brown adipose tissue (Linhart et al., 2001). Increased levels of free fatty acids and triglycerides as well as insulin sensitivity in the Fra1tg mice could also be due to the resulting reduced level of C/ebpα in adipose tissue and liver. C/EBPα has been described to be involved in hepatic glucose and lipid metabolism as it regulates the transcription of genes regulating fatty acid uptake and lipolysis, partly in cooperation with Pparγ2 (Wu et al., 1999).
These essential functions are clearly demonstrated as C/ebpα deficiency in mice leads to an early post-natal lethality due to the lack of hepatic gluconeogenesis (Wang et al., 1995). We analyzed the expression levels of genes regulating adipocyte function, and observed a reduced level of Cd36, Lpl, Plin1, Scd1 and Pnpla2 in the visceral adipose tissue of 10-week-old Fra1tg mice. However, when analyzing adipose tissue of 6-week-old mice, we did not observe reduced levels of the lipogenic gene Scd1, as well as of Pnpla2, encoding the rate-limiting enzyme regulating hydrolysis of triglycerides (Chandak et al., 2010). Therefore, alterations in the expression of these genes are most likely a result of the increased lipodystrophic phenotype observed when aging. Of particular interest is Cd36 which is down-regulated in young as well as in older Fra1tg mice. CD36, a transmembrane protein responsible for fatty acid uptake in peripheral tissues is directly regulated by C/EBPα (Qiao et al., 2008). Interestingly, CD36 deficient mice develop a similar lipodystrophic phenotype with increased fasting levels of free fatty acids and triglycerides (Febbraio et al., 1999). In addition, we observed a reduced expression level of lipoprotein lipase (Lpl) that was described to be up-regulated by C/ebpα overexpression in vitro (Olofsson et al., 2008). LPL is located in the endothelium of adipose tissue capillaries and releases postprandial lipids that
LPL enables the lipid uptake in adipose tissue and muscle (Frayn, 1998). Like the CD36 deficient mice, Lpl knockout mice show a reduced amount of white adipose tissue and an increased level of triglycerides caused by their inability to remove triglycerides from the blood (Weinstock et al., 1995).
Therefore, the phenotype of reduced lipid storage in adipocytes with increased triglyceride and free fatty acid levels in the plasma can at least in part be linked to the decreased expression of the C/EBPα-regulated lipid transporter Cd36. A possible involvement of Fra1 in lipid and glucose metabolism is also supported by studies reporting an induction of Fra1 expression after stimulation with insulin (Culbert and Tavare, 2002) or by fatty acids (Distel et al., 1992).
In addition, we showed that Fra1tg mice are completely protected from high fat diet-induced obesity including the lack of ectopic lipid storage in the liver as well as of the increase in adipocyte diameter. These features are also described in other lipodystrophic mouse models that are unable to store triglycerides, like the Crebbp+/- mouse (Yamauchi et al., 2002b), or the CD36 deficient mouse (Qiao et al., 2008).
The metabolic parameters analyzed in this study are summarized in Table 4.1.
decreased unchanged increased
serum parameter leptin,
glucose1 insulin
osteocalcin, triglycerides1, free fatty acids1 mRNA expression of
adipokines Lep, Adipoq, Tnfα Retn, Il-6 genes involved in
lipogenesis and lipolysis2
Acaca, Fasn, Scd1,
Pnpla2, Lpl Cd36, Plin1
Table 4.1 Metabolic parameters analyzed in Fra1tg mice. 1Serum levels of fasted mice and 2expression level in 10-week-old mice are shown.
4.6 Phenotype of mice with an adipocyte-specific deficiency of Fra1
Fra1 transgenic mice and Fra1 deficient mice develop a mirroring bone phenotype (Eferl et al., 2004; Jochum et al., 2000). This suggests that the effect of Fra1 on adipogenesis would not only be a consequence of the overexpression, but rather illustrating a function of Fra1 in adipogenesis in wild-type mice. In comparison, no bone or fat phenotype was reported for the FosB knockout mice (Brown et al., 1996; Gruda et al., 1996; Zenz et al., 2008). This is in agreement with the almost undetectable expression of FosB in the bone or fat of wild-type mice. In our studies, we observed a 10-fold higher expression of Fra1 compared to FosB in the adipose tissue. Thus, the role of Fra1 in adipogenesis can be distinguished from the effects of ∆FosB in vivo that might, in the absence of a forced expression not exert any role in osteoblast or adipocyte differentiation.
Nevertheless, we could not observe any difference in body weight, in the ratio fat to body weight or in adipocyte diameter in the Ap2-cre Fra1f/f mice. The fatty acid binding protein 4, Fabp4 (Ap2), is an intracellular fatty acids transport protein (Fruhbeck et al., 2001; Sun et al., 2003) and a marker for terminal adipocyte differentiation. However although only a slight expression of the recombinase was originally reported in other tissues than white and brown adipose tissue, for example in liver, heart and muscle of Ap2-cre mice (Barak et al., 2002), we observed a Ap2-cre mediated deletion of Fra1 in several tissues. The expression of the cre recombinase at a quite late stage of adipocyte differentiation, however, is probably the cause of the lack of a fat phenotype in Ap2-cre Fra1f/f mice, as C/ebpα, which seems to be the effector of Fra1, is already expressed.
4.7 Conclusions
In conclusion, we report a novel function for the AP-1 transcription factor Fra1 in regulating adipocyte differentiation. Fra1 is expressed in adipose tissue and overexpression causes a block in adipocyte differentiation in vitro and in vivo, due to a direct inhibition of C/ebpα expression (Fig. 4.1). Therefore, these findings indicate a diverging role of the different Fos family members during adipocyte differentiation, with in vivo experiments showing that one Fos family member namely ∆FosB, can lead to a lipodystrophic phenotype due to a systemic regulation of metabolism and a second one, Fra1, acting locally and cell-autonomously on the differentiation of adipocytes and osteoblasts.
Figure 4.1: Supposed function of Fra1 on adipocyte differentiation. Mesenchymal progenitor cells have the capacity to differentiate, amongst others, to osteoblasts and adipocytes. Thereby, adipocyte differentiation is regulated by the transcription factors C/ebpβ and δ, that lead to the expression of Pparγ2 and C/ebpα. We suppose Fra1 to regulate adipogenesis by inhibiting the expression C/ebpα.
This work is highly relevant for human health. Indeed, the growing incidence of obesity, leading to an increased susceptibility for type 2 diabetes and cardiovascular diseases, clearly underlines the importance of gaining detailed insight into the mechanistic basis of adipogenesis. In addition, various studies have described the systemic interaction of bone and adipose tissue. However factors locally co-regulating adipocyte and osteoblast differentiation as well as their influence on the glucose and insulin metabolism are still largely unknown. In this thesis we identified Fra1 as being one of these factors.
maturation
mesenchymal progenitor
cell
pre-adipocyte mature
adipocyte determination/
commitment differentiation
immature adipocyte C/ebpββββ
C/ebpδδδδ
Pparγγγγ2
osteoblast C/ebpαααα
Fra-1
Glut4 (insulin-stimulated glucose transport)
CD36 (lipid transport)
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