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MicroRNAs (miRNAs) are involved in a plethora of important biological processes, from embryonic development to homeostasis in adult tissues. Recently, miRNAs have emerged as a class of epigenetic regulators of metabolism and energy homeostasis. We have investigated the role of miRNAs in the regulation of adipogenic differentiation. In this article, we demonstrate that the miR-27 gene family is downregulated during adipogenic differentiation. Overexpression of miR-27 specifically inhibited adipocyte formation, without affecting myogenic differentiation. We also found that expression of miR-27 resulted in blockade of expression of PPARγ and C/EBPα, the two master regulators of adipogenesis. Importantly, expression of miR-27 was increased in fat tissue of obese mice and was regulated by hypoxia, an important extracellular stress associated with obesity. Our data strongly suggest that miR-27 represents a new class of adipogenic inhibitors and may play a role in the pathological development of obesity.
MicroRNAs (miRNAs) have emerged as an important class of post-transcriptional regulators of metabolism in several cell types, including β-cells, muscle cells, and adipocytes . They appear to be involved in diverse aspects of cellular responses to metabolic demands or stresses, from invertebrates to vertebrates. A forward genetic screening in Drosophila melanogaster provided the first example that miR-14 plays a critical role in the regulation of triacylglyceride metabolism in fruit flies . With a similar approach, miR-278 was recently identified as a potential regulator of energy metabolism in the fat body of fruit flies . In vertebrates, miR-375 and miR-376, both of which are abundantly expressed in pancreatic β-cells, are involved in the control of insulin secretion . Furthermore, the highly conserved miRNA miR-1 has been found to exert a significant influence on myogenic differentiation and muscle functions in invertebrates  as well as in mammals .
Adipose tissue functions are essential to energy metabolism because adipose tissue is not only an energy depot , but also a source of endocrine factors [8,9]. Adipocytes are derived from mesenchymal stem or progenitor cells via a lineage-specific differentiation process called adipogenesis. Adipogenic differentiation is accomplished by a cascade of three major transcriptional events characterized by the transcriptional induction of: (a) the early genes C/EBPβ and C/EBPδ; (b) the determination genes PPARγ and C/EBPα, also regarded as master regulators of adipogenesis; and (c) adipocyte-specific genes such as those encoding fatty acid synthase and fatty acid-binding proteins [10–12]. Epigenetic regulation of adipose functions mediated by miRNAs has been emerging as an important mechanism in the study of energy metabolism and obesity. By comparing miRNA profiles, Kajimoto et al.  have found differential profiles of miRNA expression between preadipocytes and mature adipocytes, suggesting a role for miRNAs in the regulation of adipogenic differentiation. Consistent with this notion, microarray analysis has identified two classes of miRNAs, miR-143 and the miR-17/92 cluster, the expression of which is moderately (two-fold to three-fold) increased during adipogenic differentiation [14,15]. Inhibition of miR-143 expression by an antisense oligonucleotide results in inhibition of adipogenesis in vitro , whereas overexpression of the miR-17/92 cluster moderately increases adipocyte formation in vitro . Although these studies have provided evidence for a role of miRNAs in adipogenesis, there is still no evidence regarding expression of miRNAs in adipose tissues, especially their regulation associated with obesity.
Adipose tissue undergoes a dramatic expansion in obesity, which eventually results in adipose tissue dysfunction. Our studies have shown that obese tissue becomes hypoxic or oxygen-deficient, and hypoxia facilitates inflammatory responses in adipocytes [16,17]. We have also shown that hypoxia strongly inhibits adipogenic differentiation [18,19]. However, it remains to be determined whether miRNAs are differentially regulated or play a role under obese conditions in vivo.
In the current study, we investigated the role of miRNAs in adipogenic differentiation using the mouse embryonic fibroblast-derived 3T3-L1 preadipocytes  and mouse bone marrow-derived OP9 mesenchymal stem/progenitor cells . We found that expression of the miR-27 family genes (miR-27a and miR-27b) was downregulated upon adipogenic differentiation. Overexpression of miR-27 resulted in robust and specific inhibition of adipogenic differentiation with blockade of PPARγ and C/EBPα expression. Importantly, miR-27 expression was elevated in adipose tissue of genetically obese ob/ob mice. We also found that the environmental stress, hypoxia, was involved in the regulation of miR-27 expression. Our data suggest that the miR-27 gene family is potentially an important class of negative regulators of adipogenesis and may play a role in the regulation of adipose functions associated with obesity.
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In this article, we have identified miR-27a and miR-27b as a new class of adipogenic regulators that strongly inhibit adipogenesis. Although the gene loci of miR-27a and miR-27b are located in different chromosomes (mouse 8 and human chromosome 19 for miR-27a; mouse chromosome 13 and human chromosome 9 for miR-27b), our data reveal a concerted downregulation of the miR-27 gene family during adipogenic differentiation of mesenchymal progenitor cells. Consistent with our observation, an independent study has found that miR-27a appears to be downregulated upon adipogenic differentiation of 3T3-L1 preadipocytes . Our evidence indicates that the inhibitory effect of miR-27 on adipogenic differentiation is specific. Both miR-27a and miR-27b inhibit adipogenic conversion of mesenchymal progenitor cells from different tissue sources, such as the bone marrow-derived OP9 cells and the embryo-derived fibroblastic 3T3-L1 cells. On the other hand, neither miR-27a nor miR-27b significantly affects myogenic differentiation. Interestingly, a very recent study has shown that downregulation of miR-27 increases intracellular lipid accumulation in hepatic stellate cells . Together, these findings suggest a role of miR-27 in multiple metabolic pathways. However, because miR-27 has the potential to target over 3000 genes, it is possible that miR-27 can regulate many other biological processes. It has been shown that miR-27a plays a role in cell cycle regulation in breast cancer cells  and facilitates the growth of gastric cancer cells . On the other hand, miR-27b has been shown to regulate the expression of cytochrome P450, a drug-metabolizing enzyme, in cancer cells . It is possible that the biological function of miR-27 is manifested in a cell type-dependent manner and/or under certain pathophysiological conditions.
As compared with other reported miRNAs that have been investigated in adipogenesis, the miR-27 genes exhibit the strongest function as a class of negative regulators of adipogenesis. Wang et al.  have shown that expression of the miR-17/92 cluster is moderately upregulated during adipogenesis. Overexpression of the miR-17/92 cluster moderately enhances adipogenic conversion but does not initiate adipogenic differentiation of mouse 3T3-L1 preadipocytes in the absence of adipogenic hormones. A moderate increase in miR-143 has also been found during the late stage (≥ 7 days) of adipogenic differentiation of human preadipocytes . Treatment with antisense oligonucleotides against miR-143 decreases lipid accumulation in adipocytes . However, Kajimoto et al.  have shown that antisense inhibition of upregulated miRNAs does not affect adipogenic differentiation of 3T3-L1 cells. These observations, nonetheless, suggest the existence of extensive crosstalk or functional overlap among different miRNA genes.
The miR-27 genes appear to inhibit adipogenesis before preadipocytes become committed to terminal differentiation. The time course study (Fig. 2) has shown that miR-27a and miR-27b are capable of blocking adipogenic differentiation when introduced before or at the start of adipogenic stimulation by IDM. After 24 h of IDM stimulation, the miR-27 genes fail to suppress adipogenesis. Because robust transcriptional induction of PPARγ and C/EBPα generally occurs within 24–48 h of adipogenic stimulation [11,12,28], our data suggest that the miR-27 genes are not capable of preventing the committed, PPARγ/C/EBPα-expressing preadipocytes from undergoing terminal differentiation. Nonetheless, our observations indicate that miR-27 genes function by blocking the transcriptional induction of PPARγ and C/EBPα or by preventing preadipocytes from entering the stage of adipogenesis determination or commitment. The transcriptional repression of PPARγ and C/EBPα appears to be specific, because C/EBPβ and C/EBPδ, which are expressed before the induction of PPARγ and C/EBPα, are unaffected by miR-27a or miR-27b.
It is predicted by bioinformatics that PPARγ mRNA contains one putative binding site for miR-27a and miR-27b in its 3′-UTR. Our data, however, show that miR-27 does not repress PPARγ expression at the protein level, the reference standard test for microRNA function, in maturing adipocytes. Because different miR-27-targeted genes have been identified in different cell types [24–27,29], these observations suggest that the target recognition by microRNAs may be context-dependent and/or cell type specific. Alternatively, miR-27 could not overcome the strong transcriptional activation of PPARγ induced by IDM. Nonetheless, our data strongly suggest that the main mechanism by which miR-27 inhibits adipogenesis is by preventing the transcriptional induction of PPARγ in preadipocytes before the adipogenic commitment stage.
The negative regulatory functions of miR-27a and miR-27b during adipogenesis prompted us to investigate whether the expression of miR-27a and miR-27b in adipose tissue is altered under pathological conditions. Using the epididymal fat tissue from the genetically obese ob/ob mice and the genetically matched lean mice, we have clearly demonstrated that the expression of both miR-27a and miR-27b is significantly increased in ob/ob mice (Fig. 5A). Although fat-derived primary stromal cells (which also contain undifferentiated progenitor cells) have approximately three-fold higher levels of miR-27a and miR-27b than primary mature adipocytes do, it is highly possible that both fat cells and stromal cells contribute to the overall increase of miR-27 in obese fat tissue, especially under stress conditions. Further investigation is warranted to clearly determine the contributions to miR-27 expression of different cell types and/or different types of cellular stresses in adipose tissue.
As compared with physiologically normal adipose tissues, obese fat tissues create dramatically different tissue microenvironments. We and others have found that obese fat tissues experience decreased tissue oxygenation or hypoxia [9,17,30]. In this study, we have found that the expression of both miR-27a and miR-27b is maintained in preadipocytes under hypoxia (Fig. 6). This result is consistent with our previous findings that hypoxia inhibits adipogenesis [18,31] and is also consistent with the finding that miR-27a expression is increased by hypoxia . However, it is worth noting that obese fat tissue becomes not only hypoxic, but also inflammatory [8,32]. Inflammatory cytokines, such as tumor necrosis factor-α, can also inhibit adipogenesis and adipocyte functions . It is highly likely that miR-27 expression in obese mice is subjected to regulation by multiple in vivo stresses. Nonetheless, our finding suggests a potential role of miR-27 in the impairment of adipose functions associated with genetic obesity.
In summary, we have identified the miR-27 genes as a new class of epigenetic regulators of adipogenesis. We have also presented the first example of obesity differentially regulating miRNA expression. The miR-27 genes may potentially play a role in the pathological progression of obesity-related diseases.