2.1. miRNA in the Regulation of White Adipogenesis
White adipose tissue (WAT) not only constitutes a significant energy depot in the human body but also secretes numerous mediators that can affect the function of distant organs. Complex interactions between different signaling pathways and transcription factors determine the process of multipotent mesenchymal stem cell (MSC) differentiation to white preadipocytes followed by growth arrest induced by contact inhibition. Next, adipogenic stimuli promote cell cycle reentry and synchronous cell division. This process depends on the induction of transcription factors, which are members of the CCAAT/enhancer-binding protein (C/EBP) family. Subsequently, C/EBPβ activates the transcription of the major transcriptional inducers of adipogenic gene expression:
C/EBPA and peroxisome-proliferator-activated receptor γ (
PPARG), as well as several other transcription factors responsible for the terminal differentiation of preadipocytes into lipid-storing mature adipocytes
[10]. Two key signaling pathways involved in MSC differentiation toward adipocytes are the Wingless (Wnt)/β-catenin and the transforming growth factor β (TGFβ)/bone morphogenetic proteins (BMP)/Smad pathways. Activated Wnt/β-catenin signaling stimulates osteogenic gene expression; however, its inhibition induces adipogenic differentiation via the induction of adipogenic-related genes, including the abovementioned
PPARG and
C/EBPA. The interaction between β-catenin and PPARγ results in β-catenin degradation with the subsequent inhibition of Wnt/β-catenin signaling and bone formation, promoting adipogenesis. Similarly, BMP/Smad signaling favors adipogenesis by increasing the expression of
PPARG, while the TGFβ/Smad pathway exerts inhibitory effects on adipogenesis
[11][12]. The proper regulation of adipocyte development and turnover assures adipose tissue homeostasis, which is seriously disturbed in the course of obesity.
The inhibition of enzymes involved in miRNA biogenesis, such as Drosha and Dicer, depresses the differentiation of human MSCs into adipocytes, which supports a role for miRNAs in adipocyte development
[13]. Subsequently, several miRNAs have been found to be regulators of human adipocyte differentiation
[4]. WAT undergoes dynamic changes to adapt to the body’s energy balance to maintain its energy storage role. In excess energy intake, WAT augments its capacity to store energy by increasing lipid accumulation and differentiation of preadipocytes to mature adipocytes. The remodeling of WAT in response to the excess of nutrients is accompanied by changes in the WAT miRNOME, with several miRNAs being up- and downregulated
[14]. Moreover, several adipocyte-selective miRNAs implicated in adipocyte proliferation and differentiation in normal-weight individuals are differentially expressed in adipose tissues of obese subjects
[8][9][15].
miRNAs are implicated in the regulation of the critical signaling pathways related to adipogenesis. For instance, miR-9-5p, by targeting the 3’UTR of Wnt3a (a Wnt ligand) and reducing its expression, inhibits Wnt/β-catenin signaling and promotes the differentiation of rat MSCs toward adipocytes
[16]. The expression of miR-9-5p is significantly increased in visceral adipose tissue (VAT) compared to subcutaneous adipose tissue (SAT) in obese patients, suggesting that the upregulation of this miRNA can be involved in the pathogenesis of obesity in humans
[9]. High serum miR-9 levels are also considered as a marker of poor prognosis in diabetic nephropathy
[17]. A similar effect exerts miR-210, which targets the T cell-specific transcription factor 7-like 2 (TCF7L2) responsible for triggering the downstream responsive genes of the Wnt pathway, as previously shown in 3T3-L1 murine preadipocytes
[18]. This miRNA was, in turn, upregulated in the SAT of obese patients, compared to the SAT of normal-weight individuals, while weight loss led to a significant decrease in its expression
[8][9]. Moreover, the expression of miR-210 was upregulated in the SAT of obese individuals with normal glucose tolerance, compared to those diagnosed with type 2 diabetes mellitus (T2DM), suggesting its protective role in the development of obesity-related adipose tissue dysfunction
[19]. However, in patients with T2DM, serum miR-210 concentration may serve as a diagnostic biomarker of diabetic retinopathy patients and may have the ability to predict disease development and severity
[20].
In turn, miR-21 was found to positively regulate adipogenesis in human adipose-derived stromal cells (hADSCs) by binding and neutralizing TGFβ1—an inhibitor of adipogenesis
[21]. The upregulation of miR-21 in the adipose tissue of obese subjects has been consistently reported in several studies
[9][22][23], while its serum levels correlate negatively with body mass index, waist circumference and insulin levels
[24]. Another miRNA targeting TGFβ/Smad is miR-199a-5p, which promotes the adipogenic differentiation of human bone marrow stromal cells (BMSCs)
[25]. Obesity is associated with increased miR-199a-5p levels in VAT and in sera
[19][26]. However, metabolically healthy obese individuals have lower miR-199a-5p expression in SAT compared to obese patients diagnosed with T2DM
[8]. The regulation of adipogenesis can also be obtained by miRNAs acting on TGFβ/Smad-independent pathways, as shown in the case of miR-143 targeting extracellular-signal-regulated kinase 5 (ERK5)
[27]. The regulatory role of this miRNA during adipogenesis depends on the differentiation stage that it acts on. If miR-143 is overexpressed during the clonal expansion stage, it inhibits the adipogenic differentiation of adipose tissue-derived stromal cells (ADSC). On the contrary, miR-143 overexpression during the growth arrest stage or terminal differentiation stage promotes adipocyte differentiation
[28]. The expression of this miRNA is upregulated in the sera of obese individuals and decreased in the adipose tissue of previously obese patients after successful weight loss
[29].
Several other miRNAs have been shown to inhibit MSC differentiation towards osteoblasts and, therefore, to promote adipogenesis. This takes place in the case of miR-204 and miR-637, which target members of the Wnt/β-catenin signaling pathway: runt-related transcription factor 2 (RUNX2) and osterix (OSX), respectively
[30][31]. The increased expression of miR-204 characterizes VAT in human obesity and impairs mitochondrial biogenesis and the development of brown adipose tissue (BAT) in rodents
[9][15]. Obesity-related changes in miR-637 expression in human adipose tissue have not been reported to date; however, its serum levels increase during dietary weight loss intervention
[32].
In turn, miR-27b and miR-130a, by interfering with PPARγ and miR-31 by targeting C/EBPα, were found to favor the osteogenic differentiation of MSCs
[33][34][35]. Moreover, miR-130a can suppress MSC differentiation towards adipocytes via interfering with adenomatosis polyposis coli downregulated 1 (
APCDD1), encoding an inhibitor of the Wnt signaling pathway
[36]. Consistently, decreased levels of this miRNA accompany human preadipocyte differentiation
[8][23][37]. However, while the decreased expression of miR-27b was found in the SAT of obese patients with T2DM and VAT, and individuals with non-alcoholic steatohepatitis (NASH), the SAT of metabolically healthy obese individuals was characterized by increased levels of miR-27b, and weight loss did not influence its expression
[9][15][22][38]. In turn, obese patients with polycystic ovary syndrome (PCOS) tended to exhibit decreased serum miR-27b levels
[39]. Data on obesity-induced changes in miR-130a are inconsistent; in some studies, obesity was associated with increased miR-130a levels in SAT and in sera, while in others, it was associated with its downregulation. However, it should be noted that while Nardelli et al. measured the expression of both miR-130a isoforms (3p and 5p), in the study by Wang et al., only the miR-130a-5p level was assessed
[15][22][40]. In turn, miR-31-5p was found to be upregulated in the VAT compared to the SAT of obese adult individuals and in the sera of obese adolescents
[9][41].
miR-181a is an example of another miRNA regulating adipogenesis by targeting PPARγ; however, miR-181a suppression decreased the expression of PPARγ in porcine primary preadipocytes
[42]. The role of this miRNA in the pathogenesis of obesity and related complications in humans is less clear. Ortega et al. found that increased miR-181a expression is a hallmark of preadipocytes originating from the SAT of obese subjects, compared to normal-weight individuals, while Kloting et al. observed its upregulated levels in the SAT of T2DM patients compared to obese subjects without metabolic complications of obesity
[8][19]. Moreover, miR-181a expression was significantly elevated in the serum of patients with non-alcoholic fatty liver disease (NAFLD), suggesting it can serve as a disease marker
[43]. However, in other studies, miR-181a expression was downregulated in the SAT of metabolically healthy obese patients and VAT of obese individuals with NASH
[38][44].
In addition to the direct interaction with PPARγ mRNA, miRNA can regulate adipogenesis by targeting proteins involved in the regulation of PPARγ activity. For instance, miR-146b has been found to promote preadipocyte differentiation via interaction with Sirt1, the NAD-dependent deacetylase, known as a PPARγ inhibitor
[45]. Human obesity is associated with the upregulated expression of miR-146b in SAT and sera, which significantly decreases after weight loss
[9][46][47]. Interestingly, a high miR-146b serum concentration is a T2DM predictor in obese adolescents, while lower levels are observed exclusively in the VAT of patients with NASH and pericellular fibrosis but are not changed between NASH and non-NASH NAFLD patients
[38][47].
The decreased expression of
SIRT1 correlates negatively with the expression of several other miRNAs in the adipose tissues of obese patients, including those involved in pro-inflammatory responses (miR-22-3p), the inhibition of adipose tissue browning (miR-34a-5p) and the activation of white adipogenesis (miR-181a-3p), suggesting that the interaction between miRNAs and SIRT1 constitutes a critical regulatory mechanism in adipocyte homeostasis
[42][44][48][49].
Given that a single miRNA targets several mRNAs, its regulatory influence on adipocyte differentiation can be exerted by triggering different cellular pathways. For instance, in different experimental conditions, miR-103 was found to promote adipogenesis via (i) targeting retinoic acid-induced protein 14 (RAI14) in the early stages of adipogenesis; (ii) activation of the protein kinase B/mammalian target of a rapamycin signaling pathway (AKT/mTOR pathway); and (iii) reversing the anti-adipogenic effects of myocyte enhancer factor 2D (MEFD2), which is a transcription factor that negatively regulates preadipocyte differentiation by downregulating the expression of multiple adipocyte markers (e.g., PPARγ and C/EBPα)
[50][51]. However, in humans, obesity seems to have little influence on miR-103 expression in adipose tissue, and thus its isoform, miR-103a-3p, is even recommended as a reference for the analysis of miRNA expression in adipocytes
[52]. On the contrary, miR-103, together with miR-107, can promote endoplasmic reticulum stress-mediated apoptosis in murine preadipocytes by targeting the Wnt3a/β-catenin/activating transcription factor 6 (ATF6) signaling pathway
[53]. The expression of miR-107 was downregulated in the SAT of obese individuals compared to normal-weight subjects and in the VAT of those diagnosed with NASH
[22][38][54]. Moreover, miR-107 SAT levels decreased after bariatric surgery, suggesting persistent, obesity-related adipose tissue dysfunction
[9]. In turn, elevated miR-103/miR-107 serum levels are a predictor of insulin resistance in obese adolescents
[55].
For many years, BAT has been thought to play a marginal role in adult energy homeostasis. However, recent research has increased our understanding of the mechanisms involved in the development and activation of brown adipocytes and their contribution to metabolic health. It has been revealed that miRNAs also play a significant role in regulating these processes.