The ability to express UCP1 in WAT depots is associated with resistance to obesity, as shown in genetic studies with various mouse strains as well as overexpression studies (1
). In the latter case, simply overexpressing UCP1 in WAT appears sufficient to promote mitochondrial biogenesis (49
). Recently, several reports have indicated that mitochondrially derived signals such as those that stem from changes in mitochondrial membrane potential or ATP production can promote mitochondrial biogenesis by a process known as “retrograde signaling,” but the molecular basis of this is not clear (31
). Therefore, because of the evidence for decreased mitochondrial function in metabolic disease (34
) and the search for mechanisms to combat obesity, understanding the molecular basis of brown versus white adipocyte differentiation continues to be of interest.
What factors and pathways control the differentiation of brown versus white adipocytes has been a long-standing question (22
). More recently, there has been a debate in the field, based on several compelling observations, as to whether brown and white adipocytes have the capacity to interchangeably “transdifferentiate” between these two cytotypes (10
). The SNS is the main driver for the development and maintenance of the brown adipocyte phenotype. It is well established that under conditions in which SNS activity is minimized, such as when animals are maintained at thermoneutrality, brown adipocytes lose their characteristic features of UCP1 expression and their high density of mitochondria (9
). If maintained in these conditions long enough, these cells can begin to produce typical white adipocyte markers, such as leptin (3
). When SNS function is reestablished, expression of UCP1 and mitochondrial biogenesis is restored. Therefore, brown adipocytes might be able to morphologically resemble white adipocytes but retain a genetic “memory” that allows them to reacquire the brown adipocyte phenotype when stimulated.
In the pursuit of understanding the molecular basis for the brown versus white fat fate, investigators have searched for the pathways and factors that activate or promote brown adipocyte differentiation. For example, PGC-1α was discovered as a factor that was highly enriched in brown but not white adipocytes (41
) and now appears to be important for mitochondrial biogenesis per se in many cell types (16
). Another molecule called prdm16 has recently been proposed as a determining factor for brown adipocyte differentiation (51
), but its mechanism is unclear. In addition to these approaches, it is equally useful to identify those elements that actively inhibit or repress brown adipocyte differentiation or its thermogenic activity. In that regard, targeted disruption in mice of several genes directly involved in energy storage and fat accumulation have been shown to lead to a lean phenotype and dramatic increases in UCP1 expression in adipocytes, particularly in white fat depots (see reference 20
for a review). For example, the apoptotic family member CIDE-A is highly expressed in brown fat, where it appears to interact with UCP1 to suppress its uncoupling function. In the absence of CIDE-A, there is unrestrained uncoupling of respiration, and the animals exhibit a lean phenotype (61
). There are also several nuclear transcription factors whose absence also leads to a similar phenotype: these include the LXRs, ERRα, retinoblastoma protein, and RIP140 (20
In the case of the LXRs, they normally promote energy storage through the expression of the lipogenic transcription factor SREBP1c, while the genetic absence of both subtypes of LXR leads to ectopic expression of UCP1 in muscle and WAT, which is more pronounced following consumption of a fat- and cholesterol-containing “Western” diet (26
). Here in this study we show that, unlike what was found in the study of Kalaany et al., mice lacking only LXRα have a significantly higher basal body temperature that is consistent with the greater levels of expression of UCP1 in both BAT and WAT. LXRα−/−
mice also possess more mitochondria and express a broad range of genes that encode mitochondrial proteins and specific markers of brown adipocytes. Thus, it would seem that in adipose tissue LXRα functions as an inhibitor of the common SNS and βAR signaling pathways that promote both diet-induced and cold-induced thermogenesis. Since the results for mice and cultured fat cells lacking LXRα alone were indistinguishable from results obtained with LXR double knock-out samples, this strongly suggests that LXRα is the operationally important isoform for this phenotype of increased basal UCP1 expression and mitochondrial biogenesis.
In the current work, we demonstrate by a number of different approaches that agonist-activated LXRα directly antagonizes transcription of the Ucp1 gene through a DR-4 element in the Ucp1 gene promoter to block the induction that results from SNS activation. First, using several different adipocyte models, we observed that the suppression of Ucp1 gene expression was evident only when both LXRα and its selective agonist were present. Adipocytes differentiated from WT and LXRα−/− MEFs showed that cAMP-stimulated UCP1 expression could be blocked only in WT cells and not in LXRα-null cells. An important point is that LXRα−/− adipocytes expressed significantly more UCP1 even under unstimulated basal conditions, and there was an enormous increase in expression in response to FSK or the βAR agonist Iso. Importantly, we determined that cultured adipocytes from the LXRα−/− mice had higher mitochondrial density, oxygen consumption, and uncoupled respiration, which was also reflected in the increased expression of a panel of mitochondrial and brown adipocyte marker genes in these cells.
An important factor shown to promote Ucp1 expression in brown adipocytes, and mitochondriogenesis in general, is the coactivator PGC-1α. PGC-1α participates together with PPARγ in Ucp1 gene transcription in response to β-adrenergic stimulation, through a cAMP/p38 MAPK pathway (15
). Transcription of both the Ucp1 and PGC-1α genes in response to β-adrenergic stimulation often shows close temporal and mechanistic parallels. However, the inhibitory effect of LXRα is specifically on the Ucp1 gene and not a primary consequence of changes in PGC-1α itself. Instead, we identified a DR-4 element in the Ucp1 gene enhancer to which LXRα binds. As shown in ChIP assays, this site is immediately adjacent to the DR-1 site that becomes occupied by PPARγ upon elevations in cAMP (6
). Curiously, we observed that conditions that elevate cAMP also result in LXRα being recruited to the enhancer, even in the absence of its ligand. It is unclear whether LXRα is chemically modified in some way and what the function of this cAMP-dependent binding of LXRα is for the enhancer, since the genetic absence of LXR obviously does not hamper the ability to transactivate the Ucp1 gene; instead, the expression of Ucp1 is enormously increased. In any event, the significant point is that upon agonist activation of LXRα, the cAMP-induced binding of PPARγ is lost. This suggests that the dismissal of PPARγ is involved in the suppression of Ucp1 transcription. Other interpretations were considered. These included a possible direct interaction between LXRα and PPARγ, but this is unlikely for two reasons. First, in response to cAMP, which is the stimulus for Ucp1 transcriptional activation, LXRα and PPARγ are nevertheless both found on the Ucp1
enhancer. Second, reports of cross talk between PPAR and LXR have appeared and, at least with cell culture models of forced overexpression, show that these two nuclear receptors can interfere with each other's ability to activate target genes by competing for limiting amounts of RXR (23
). However, for our studies, this explanation is unlikely, since the provision of excess receptors did not eliminate the ability of LXR to repress Ucp1 gene expression. Finally, it is known that thyroid hormone augments the process of nonshivering thermogenesis and UCP1 expression in BAT (55
), but the exact mechanism is still not clear. Although thyroid hormone receptors can bind DR-4 elements (21
), we can find no support for an involvement of a thyroid hormone receptor from either gene transfection or in vitro DNA-protein binding experiments that would explain the DR4-dependent regulation of Ucp1 gene expression (data not shown).
Since the control of gene expression by nuclear receptors requires the recruitment of coregulator complexes, RIP140 was a good candidate to investigate for several reasons. The phenotype of RIP140-null mice is remarkably similar to that of LXRα−/−
). These animals are reported to be lean and resistant to obesity induced by a high-fat diet, due to a higher mitochondrial uncoupling-derived energy expenditure. RIP140 is also highly induced during white adipocyte differentiation (39
). Interestingly, this is driven by the nuclear receptor ERRα, the absence of which in mice has also been reported to produce a hypermetabolic, lean mouse (32
). Finally, RIP140 was previously found on the Ucp1
), although there was no particular association with any nuclear receptor. Recently, Debevec et al. proposed that RIP140 regulates Ucp1 gene transcription with the involvement of ERRα and PPARα (14
), but no specific binding site was proposed. We clearly show by ChIP assays that RIP140 appears on the Ucp1
promoter only in response to the agonist activation of LXR, when PPARγ is eliminated from the promoter. In addition, RNAi silencing of RIP140 expression eliminated the ability of the LXR agonist to suppress Ucp1 expression; results for RIP140 RNAi in LXRα−/−
cells were similar. Other corepressors, such as nuclear receptor corepressor and the silencing mediator of retinoic acid and thyroid hormone receptors, might be considered to play a role in the repression mediated by LXRα, since they have been shown in another system to be involved in LXR ligand-independent transcriptional repression at the SREBP1 promoter (59
). However, repression of the Ucp1
promoter by LXRα occurs only in response to cAMP and in the presence of the LXRα ligand. Thus, with the discovery of the DR-4 in the Ucp1
enhancer that mediates this LXRα repression, there is clearly a need for further work to gain a better mechanistic understanding of the complex molecular interactions that are taking place on this functionally “crowded” enhancer region of the Ucp1
In summary, our results show a novel role for LXRα to function as a direct transcriptional repressor. It also suggests that LXRα is functioning as a “brake” on the expression of genes that promote the differentiation process toward the brown adipocyte phenotype. It is unclear at this point whether the molecular basis of this phenomenon is due to (i) the ability of LXRα to repress a network of genes that contribute to the brown adipocyte phenotype or (ii) the robust expression of UCP1, now unrestrained by that lack of LXRα, creating a mitochondrially derived signal that promotes mitochondrial biogenesis by retrograde signaling. Both mechanisms are plausible, and this question will need to be addressed in future studies. On a physiological level, LXRα is equally abundant in both BAT and WAT, but UCP1 and a rich density of mitochondria are found mainly in BAT. This raises the question as to whether the pivotal regulatory factor by which LXRα regulates Ucp1 and mitochondrial biogenesis involves the availability of an LXR ligand. Of the enzymes that are presently known to be involved in the synthesis of oxysterols, cholesterol 24-hydroxylase is confined to the central nervous system, leaving cholesterol 25-hydroxylase (CH25H) as a relevant enzyme expressed in adipose tissue, although the exact nature of the true endogenous ligands are still a matter of debate (D. Russell, personal communication). Interestingly, when expression levels of CH25H were measured, there was 30-fold more CH25H in WAT than in BAT (http://www.thehamner.org/docs/collins-mcb08.pdf
). These observations would appear to suggest that under physiological conditions when conditions favor the storage of lipid-rich energy, it would be preferable for a system such as that we have shown for LXRα to suppress energy-consuming events such as UCP1-dependent uncoupling and wasting of energy through thermogenesis. Altogether, this study provides an interesting new avenue of investigation into understanding the signals and mechanisms that control the differentially expressed genes in white and brown adipocytes.