Adipose Tissue MicroRNAs as Regulators of CCL2 in Obesity
In this study, we identified a set of miRNAs uniformly expressed in human WAT that are altered in obesity and that regulate CCL2 secretion from human fat cells and macrophages. CCL2 is a key factor involved in the development of obesity-induced inflammation. Our findings show that miRNAs regulate CCL2 production via both direct and indirect mechanisms, the latter involving a network of distinct TFs.
miRNAs have emerged as important transcriptional regulators in both physiological and pathophysiological conditions. A number of studies identify several miRNAs to be dysregulated in obese WAT. However, there has been only a modest overlap concerning the specific miRNAs that have been described in each study. Our present results defining a set of 11 adipocyte-specific miRNAs downregulated in obesity were found in the largest cohort of subjects published so far (n = 56) and performed in both WAT and isolated adipocytes. Admittedly, our candidates only partially overlap with the previous studies, possibly explained by differences in study designs, for example, the use of only limited numbers of lean subjects, the inclusion of patients with diabetes, and/or the use of different technical methodologies.
MARA allowed us to construct a transcriptional regulatory network present in adipocytes and altered by obesity that could mediate the effects of individual miRNAs on CCL2 secretion. The predicted network comprised several TFs with well-characterized roles in obesity-induced inflammation and five miRNAs that were directly or indirectly linked to CCL2. For two of the miRNAs, we were able to confirm the regulatory pathways proposed by our model. In particular, we demonstrated a signal circuit from miR-126 directly to CCL2 and from miR-193b to CCL2 either via ETS1 as a single TF step or via a series of other TFs (MAX, NFKB1, RELB, and STAT6). These findings were corroborated by several independent validation experiments and demonstrate a hitherto unknown role for miRNAs in the regulation of adipocyte CCL2 production. miRNA-193b may be of particular importance since its expression was associated with CCL2 secretion from subcutaneous WAT, independent of BMI. To our knowledge, several miRNAs identified in this study have not been previously associated with adipocyte function (miR-26a, −92a, −126, −145, −652, and let-7a/d) or controlling inflammatory pathways (miR-92a, −193a/b, and −652). Moreover, in recent murine studies, let-7 and miR-193b were implicated in controlling muscle glucose metabolism and brown fat differentiation, respectively. This further supports a role for miRNAs in regulating different aspects of metabolism.
In this study, we used primary cultures of in vitro differentiated human adipocytes and the human monocyte/macrophage cell line (THP1) in our functional experiments. It could be argued that freshly isolated mature fat cells and adipose-derived macrophages should have been investigated. However, recent publications show that purified mature fat cells are not ideal for studies on inflammation since the isolation procedure per se affects inflammatory pathways. In addition, for both practical and ethical reasons, it is not possible to obtain sufficient amounts of adipose-derived macrophages from human biopsies.
Using the present approach, we were not able to identify how 8 out of 10 candidate miRNAs affect CCL2 secretion. There could be several explanations for this. Most important, the network constructed using the present approach is not complete. For instance, TFs without known DNA-binding motifs and other regulatory proteins (i.e., transcriptional cofactors) are not included in the current model. Therefore, more complex transcriptional regulatory networks, which include these and other factors, need to be constructed in future studies. Furthermore, we examined only subcutaneous WAT obtained from female donors and although unlikely, we cannot exclude sex- and region-specific differences in the regulation of CCL2 production.
Although the focus of this study was on fat cells, our data in THP1 cells suggest that miR-193b and miR-126 may also be important for the regulation of CCL2 secretion in macrophages. However, whether the signaling circuit established in adipocytes is also present in macrophages remains to be determined. In any case, the clinical relevance of our findings is supported by the observation that there was a strong association between the expression of miR-193b or miR-126 and the mRNA levels of the M1 macrophage marker ITGAX, independent of BMI.
While CCL2 may induce insulin resistance in skeletal muscle cells via direct mechanisms, there are no reports of such effects in fat cells. This suggests that the link between CCL2, adipose tissue inflammation, and local insulin resistance is indirect. Hence, CCL2 may initiate and possibly sustain migration of proinflammatory cells into WAT, which in turn (together with the adipocytes) secrete a number of proteins (e.g., tumor necrosis factor-α and IL-6), which affect insulin signaling directly or indirectly, as discussed previously.
Taken together, our results suggest that specific miRNAs may be important regulators of inflammation in human adipose tissue through their effects on CCL2 secretion from adipocytes and macrophages. This may be mediated by direct interactions with CCL2 mRNA and/or via indirect effects on TF circuits. miRNAs could therefore constitute novel potential targets for treating insulin resistance and type 2 diabetes in obesity. The approach presented herein may form the basis for future studies focusing on complex interactions regulating other aspects of human adipose tissue function.
Discussion
In this study, we identified a set of miRNAs uniformly expressed in human WAT that are altered in obesity and that regulate CCL2 secretion from human fat cells and macrophages. CCL2 is a key factor involved in the development of obesity-induced inflammation. Our findings show that miRNAs regulate CCL2 production via both direct and indirect mechanisms, the latter involving a network of distinct TFs.
miRNAs have emerged as important transcriptional regulators in both physiological and pathophysiological conditions. A number of studies identify several miRNAs to be dysregulated in obese WAT. However, there has been only a modest overlap concerning the specific miRNAs that have been described in each study. Our present results defining a set of 11 adipocyte-specific miRNAs downregulated in obesity were found in the largest cohort of subjects published so far (n = 56) and performed in both WAT and isolated adipocytes. Admittedly, our candidates only partially overlap with the previous studies, possibly explained by differences in study designs, for example, the use of only limited numbers of lean subjects, the inclusion of patients with diabetes, and/or the use of different technical methodologies.
MARA allowed us to construct a transcriptional regulatory network present in adipocytes and altered by obesity that could mediate the effects of individual miRNAs on CCL2 secretion. The predicted network comprised several TFs with well-characterized roles in obesity-induced inflammation and five miRNAs that were directly or indirectly linked to CCL2. For two of the miRNAs, we were able to confirm the regulatory pathways proposed by our model. In particular, we demonstrated a signal circuit from miR-126 directly to CCL2 and from miR-193b to CCL2 either via ETS1 as a single TF step or via a series of other TFs (MAX, NFKB1, RELB, and STAT6). These findings were corroborated by several independent validation experiments and demonstrate a hitherto unknown role for miRNAs in the regulation of adipocyte CCL2 production. miRNA-193b may be of particular importance since its expression was associated with CCL2 secretion from subcutaneous WAT, independent of BMI. To our knowledge, several miRNAs identified in this study have not been previously associated with adipocyte function (miR-26a, −92a, −126, −145, −652, and let-7a/d) or controlling inflammatory pathways (miR-92a, −193a/b, and −652). Moreover, in recent murine studies, let-7 and miR-193b were implicated in controlling muscle glucose metabolism and brown fat differentiation, respectively. This further supports a role for miRNAs in regulating different aspects of metabolism.
In this study, we used primary cultures of in vitro differentiated human adipocytes and the human monocyte/macrophage cell line (THP1) in our functional experiments. It could be argued that freshly isolated mature fat cells and adipose-derived macrophages should have been investigated. However, recent publications show that purified mature fat cells are not ideal for studies on inflammation since the isolation procedure per se affects inflammatory pathways. In addition, for both practical and ethical reasons, it is not possible to obtain sufficient amounts of adipose-derived macrophages from human biopsies.
Using the present approach, we were not able to identify how 8 out of 10 candidate miRNAs affect CCL2 secretion. There could be several explanations for this. Most important, the network constructed using the present approach is not complete. For instance, TFs without known DNA-binding motifs and other regulatory proteins (i.e., transcriptional cofactors) are not included in the current model. Therefore, more complex transcriptional regulatory networks, which include these and other factors, need to be constructed in future studies. Furthermore, we examined only subcutaneous WAT obtained from female donors and although unlikely, we cannot exclude sex- and region-specific differences in the regulation of CCL2 production.
Although the focus of this study was on fat cells, our data in THP1 cells suggest that miR-193b and miR-126 may also be important for the regulation of CCL2 secretion in macrophages. However, whether the signaling circuit established in adipocytes is also present in macrophages remains to be determined. In any case, the clinical relevance of our findings is supported by the observation that there was a strong association between the expression of miR-193b or miR-126 and the mRNA levels of the M1 macrophage marker ITGAX, independent of BMI.
While CCL2 may induce insulin resistance in skeletal muscle cells via direct mechanisms, there are no reports of such effects in fat cells. This suggests that the link between CCL2, adipose tissue inflammation, and local insulin resistance is indirect. Hence, CCL2 may initiate and possibly sustain migration of proinflammatory cells into WAT, which in turn (together with the adipocytes) secrete a number of proteins (e.g., tumor necrosis factor-α and IL-6), which affect insulin signaling directly or indirectly, as discussed previously.
Taken together, our results suggest that specific miRNAs may be important regulators of inflammation in human adipose tissue through their effects on CCL2 secretion from adipocytes and macrophages. This may be mediated by direct interactions with CCL2 mRNA and/or via indirect effects on TF circuits. miRNAs could therefore constitute novel potential targets for treating insulin resistance and type 2 diabetes in obesity. The approach presented herein may form the basis for future studies focusing on complex interactions regulating other aspects of human adipose tissue function.
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