Research Support: HH is funded by a Health Research Board Clinical Research Fellowship. Institutional support is provided by NBCRI.
Dr N Miller, Department of Surgery, National University of Ireland, Galway Clinical Science Institute, Galway, Ireland. E-mail: firstname.lastname@example.org
Obesity and the metabolic syndrome are major public health concerns, and present a formidable therapeutic challenge. Many patients remain recalcitrant to conventional lifestyle changes and medical therapies. Bariatric surgery has made laudable progress in the treatment of obesity and its related metabolic disorders, yet carries inherent risks. Unravelling the molecular mechanisms of metabolic disorders is essential in order to develop novel, valid therapeutic strategies. Mi(cro)RNAs play important regulatory roles in a variety of biological processes including adipocyte differentiation, metabolic integration, insulin resistance and appetite regulation. Investigation of these molecules and their genetic targets may potentially identify new pathways involved in complex metabolic disease processes, improving our understanding of metabolic disorders and influence future approaches to the treatment of obesity. This review discusses the role of miRNAs in obesity and related components of the metabolic syndrome, and highlights the potential of using miRNAs as novel biomarkers and therapeutic targets for these diseases.
Obesity and the metabolic syndrome are major public health concerns, and present a formidable therapeutic challenge. The incidence of this disease spectrum continues to rise and contributes significantly to global morbidity, mortality and socioeconomic burden. Current treatment modalities include lifestyle modification, diet and pharmacologic agents yet many patients remain recalcitrant to conventional medical therapy. Bariatric surgery has made laudable progress in the treatment of obesity and its related metabolic disorders, yet carries inherent risks. Scientists and clinicians must focus on improving understanding of the molecular mechanisms underpinning metabolic disorders in order to develop novel, valid therapeutic strategies. Mi(cro)RNAs play important regulatory roles in a variety of biological processes including adipocyte differentiation, metabolic integration, insulin resistance and appetite regulation (1) (Fig. 1). Investigation of these tiny molecules and their genetic targets may potentially identify new pathways involved in complex metabolic disease processes, improving our understanding of metabolic disorders and influence future approaches to the treatment of obesity.
The purpose of this review is to discuss the role of miRNAs in obesity and related components of the metabolic syndrome, and to highlight the potential of using miRNAs as novel biomarkers and therapeutic targets for these diseases.
Mi(cro)RNAs are a class of non-coding endogenous RNA molecules, only 18–25 nucleotides in length. Since their discovery in 1993, these molecules have been shown to play critical regulatory roles in a wide range of biological and pathological processes (Table 1 & Supporting information, Table S1). The definitive hypothesis of their mechanisms of action remains to be elucidated. Laterally, it has been demonstrated that miRNAs may regulate cellular gene expression at the transcriptional or post-transcriptional level, by suppressing translation of protein coding genes, or cleaving target mRNAs to induce their degradation, through imperfect pairing with target mRNAs (2). MiRNA biogenesis in the human cell is a complex process (Fig. 2) (3). The miRNA target region critical to their recognition is located at the 5′ end of the mature miRNA sequence, from bases 2 to 8, referred to as the ‘seed sequence’(4). Computational target prediction algorithms have been developed to identify putative mRNA targets, and thus place considerable importance on this seed sequence, using it to search for complementary sequences in the 3′-UTRs of known genes that exhibit conservation across species. These algorithms predict that each miRNA may potentially bind to as many as 200 targets and estimate that miRNAs control the expression of at least one-third of human mRNAs, further highlighting their crucial role as regulators of gene expression (5).
Table 1. MicroRNAs with altered expression in obesity and the metabolic syndrome
Currently 8273 mature miRNA sequences have been described in primates, rodents, birds, fish, worms, flies, plants and viruses (6). In the human genome, over 600 mature miRNAs have been reported to date; however, computational prediction estimates that this could increase to more than 1000 (7). Thus, the microRNA story is in the embryonic stage of expansion.
MiRNAs and glucose homeostasis
Maintaining appropriate blood glucose levels depends on the fine regulation of insulin release. Recently heralded as ‘ribo-regulators’ of glucose homeostasis, miRNAs play a principal role in the production and secretion of insulin, while simultaneously influencing the sensitivity or resistance of its target tissues (8–10).
The pancreatic islet-specific miR-375 plays a key role in blood glucose homeostasis through its regulation of beta cell function, particularly exocytosis of insulin-containing vesicles. Additionally, miR-124a and let-7b, both of which are also abundantly expressed in pancreatic islet cells are postulated to be important ribo-regulators of blood glucose (11). They concomitantly repress myotrophin, indicating that multiple miRNAs are likely to converge to affect translational control of a single target protein.
Tang et al. have shown that miR-30d influences insulin transcription (12). Using the pancreatic beta-cell line MIN6, miR-30d was found to be up-regulated by increased cell exposure to higher glucose concentrations, and that higher miR-30d levels were associated with increased insulin gene expression. Conversely, inhibition of miR-30d was shown to rescind glucose-stimulated insulin gene transcription. Based on these results, it is suggested that the putative target genes of miR-30d may be negative regulators of insulin gene expression. Thus, emerging data lead us to believe that the powerful miRNA regulatory mechanism is intimately involved in glucose homeostasis.
MiRNAs and diabetes
Since their discovery, miRNAs have been implicated as novel protagonists in the pathogenesis of diabetes, regulating insulin production, secretion and action (13). They also appear to play a role in the development of diabeticcomplications such as nephropathy and cardiac hypertrophy (13,14). Evidence supporting the importance of miRNAs in the pathogenesis and progression of diabetes is burgeoning. Poy et al. originally identified several miRNAs which were differentially expressed in pancreatic endocrine cell lines. MiR-375 overexpression was found to reduce beta cell number and viability and thereby suppress glucose-stimulated insulin secretion. Conversely functional experiments showed that miR-375 inhibition enhanced insulin secretion. These effects were shown to be mediated through miR-375's gene targets which include myotrophin and PDK1 (15,16) and the results indicate that miR-375 is a potentially important modulator of beta cell function.
Several proteins controlling insulin exocytosis have been identified; however, the factors regulating individual components of the secretory mechanism of beta-cells remain largely unknown. MiRNA research is now beginning to unearth, at least in part, some novel regulatory mechanisms central to this process. MiR-9 has been shown to regulate insulin release by decreasing expression of the transcription factor Onecut-2, that in turn promotes granuphilin/Slp4, a negative regulator of secretin (17).
MiR-124a expression in beta cells, found to be increased at gestational age e18.5 compared with e14.5 in mice, is postulated to be crucial to pancreatic development and differentiation of pancreatic beta cells (18). In this study using pancreatic beta cell lines, Baroukh and colleagues demonstrated that miR-124a preferentially targets Foxa2 – a master regulator of pancreatic development and of genes involved in glucose metabolism and insulin secretion (Kir6.2 and Sur-1). They conclude that this miRNA is an important regulator of a key transcriptional protein network in beta-cells, and is responsible for modulating intracellular signalling.
MiRNAs and adipogenesis
Adipose tissue is not only a storage depot of triglycerides but also has a functional role in regulating energy homeostasis. However, it is well acknowledged that abnormal and excessive fat accumulation in obese patients is associated with adverse health outcomes including an increased risk of life threatening diseases such as Type II Diabetes Mellitus, cardiovascular and cerebrovascular disease, and malignancy (19). Crucial to the development of novel therapeutic strategies for obesity, and its associated metabolic syndromes, is a better understanding of the regulation of adipogenesis. While it is accepted that this complex process is tightly controlled by a combination of multiple transcription factors and extracellular hormones, little is known about the precise mechanisms of adipogenesis. Recently miRNAs have been recognized as a class of epigenetic regulators of metabolism and energy homeostasis, primarily because the simultaneous regulation of a large number of target genes can be accomplished by a single miRNA. Emerging evidence suggests microRNAs play a key role in the pathological development of obesity by affecting adipocyte differentiation (20–22).
Existing data demonstrate that miR-14 and miR-278 in the body fat of Drosphila flies regulate lipid metabolism (23,24), miR-122 in mouse liver controls triglyceride metabolism and cholesterol biosynthesis (25). Similarly, experimental in vivo studies using antisense oligonucleotides transfected into human preadipocytes suggest that miR-143 is involved in adipocyte differentiation (21).
Takanabe et al. observed miR-143 expression to be increased 3.3-fold in adipose tissue of obese mice, and they also report similarly altered levels of the adipocyte differentiation markers PPARγ and aP2 (26). Klöting et al. carried out the first miRNA expression profiling in human omental and subcutaneous adipose tissue and uncovered significant correlations between the expression of several fat depot specific miRNAs, adipose tissue morphology and key metabolic parameters such as BMI, lipid and hormone levels (27)
Wang et al. reported that the miR-17-92 cluster is up-regulated twofold during the early clonal expansion stage of adipogenesis and that this family of miRNAs accelerate adipocyte differentiation by negatively regulating the key cell cycle regulator and tumour suppressor gene Rb2/p130 (28).
Xie et al. have recently provided the first experimental evidence for miR-103 function in adipose biology (22). Using 3T3-L1cells (Mouse embryonic fibroblast – adipose like cell line), they demonstrated that expression of miR-103 was induced approximately ninefold during adipogenesis and consequently down-regulated in adipose tissue harvested from obese mice. The accelerated miR-103 differentiation during adipogenesis was accompanied by:
• increased expression of key transcription factors (Pparγ2);
• increased levels of molecules associated with lipid metabolism (Fabp4);
• increased levels of molecules associated with glucose homeostasis (Glut4);
• increased levels of molecules associated with endocrine function of adipocytes (adiponectin).
Computational studies predict that miR-103 affects multiple mRNA targets in pathways that involve cellular acetyl-CoA and lipid metabolism (TargetScan v4.2; an online target prediction program [http://www.targetscan.org] that predicts biological targets of miRNAs by identifying the presence of conserved 8-mer and 7-mer sites that match the seed region of each miRNA) (29). The inverse pattern of miRNA expression observed in differentiating adipocytes and obese tissue indicates that obesity leads to a loss of miRNAs that characterize fully differentiated and metabolically active adipocytes. Xie et al. postulate that these changes are likely due to the chronic inflammatory environment in obese adipose tissue, which has been well described previously (22,30). The authors then show that when differentiated 3T3-L1 adipocytes were treated with TNF-α (a macrophage produced cytokine involved in chronic inflammation, largely responsible for inducing insulin resistance in obese adipose tissue) for 24 h, levels of miR-103 and miR-143 reduced in the adipocytes, while levels of miR-221 and miR-222 were increased. They also observed similar miRNA expression patterns in adipose tissue from obese mice, as well as simultaneously increased levels of TNF-α(22). They concluded that these changes in miRNA expression observed in adipocytes were likely caused by the enhanced expression of TNF-α seen in obese fat tissue.
MiRNAs and neural factors promoting obesity
The brain, central and peripheral nervous systems have been implicated as key regulators of appetite, body fat content and glucose metabolism (31). Pardini et al. provide evidence that insulin circulates at levels proportionate to body fat mass, that circulating insulin is transported to the brain, and that insulin receptors are concentrated in brain areas involved in the control of food intake and autonomic function (32).
MiRNAs have recently been shown to be differentially expressed in brain tissue and have been linked to the regulation of neural factors specific to obesity, in particular the control of appetite, and in neural signalling to liver, muscle, pancreas and gastrointestinal tract, to influence metabolism. MiR-132 has been shown to be highly expressed in brain tissue and neuronal cell types, and evidence exists to show that miR-132 is involved in the regulation of cAMP response element-binding protein (CREB) which is also known to function in glucose homeostasis (33). Moreover, several miRNAs are commonly overexpressed in both brain and pancreatic beta cells suggesting an overlap in function (e.g. miR-9, miR-124a). Plaisance et al. showed that overexpression of miR-9 (previously thought to be a brain specific miRNA) in insulin secreting cells caused a reduction in insulin exocytosis by diminishing the expression of the transcription factor Onecut-2 and, in turn, by increasing the level of Granuphilin/Slp4, a Rab GTPase effector associated with β-cell secretory granules that exerts a negative control on insulin release (17). MiR-124a similarly was initially found to be overexpressed in brain and neural tissue, and subsequently has been found to be abundant in pancreatic beta cells (13,18,34). Further evidence to support miR-124a in pancreatic function lies in the knowledge that one of the established target genes for miR-124a is FoxA2 (forkhead box protein A2, also known as HNF3 beta), a transcription factor important for beta-cell differentiation, pancreatic development, glucose metabolism and insulin secretion (35).
Hypothalamic brain-derived neurotrophic factor (BDNF) is a key element in the regulation of energy balance and has been implicated in the development of obesity (36). This protein, encoded by the BDNF gene and secreted from the hypothalamus, is a member of the neurotrophin family of growth receptors and low expression levels have previously been linked to increased appetite and obesity (37). Han et al. studied children and adults with the rare genetic condition – WAGR syndrome (Wilms' tumor, aniridia, genitourinary anomalies and mental retardation) and found that many of the people with this syndrome (68%) lack the BDNF gene, and have correspondingly low blood levels of the protein. Consequently the BDNF deficient patients had unusually large appetite and a strong tendency towards obesity. Further support for the role of BDNF in appetite regulation was provided by Stanek et al.(38) who showed that serum BDNF levels inversely correlated with appetite and weight in otherwise healthy adults. Interestingly, evidence has emerged to implicate microRNAs in the regulation of brain BDNF secretion and action. Pyramidal neurons, the primary source of BDNF in cerebral cortex, express high levels of DICER, an RNAse III endoribonuclease and key molecule for miRNA biogenesis, as well as components of the RNA-induced silencing complex (RISC), such as eIF2c which is involved in the binding of a miRNA to its target mRNA (39,40). Both miR-30a-5p and miR-195 have been shown to target specific sequences surrounding the proximal polyadenylation site within the BDNF 3′-untranslated region on chromosome 11p13. Furthermore, neuronal overexpression of miR-30a-5p and miR-195, miRNAs enriched in layer III pyramidal neurons, resulted in down-regulation of BDNF protein.
MiRNAs and liver biosynthesis of cholesterol
MiR-122 is a liver-specific miRNA implicated in cholesterol and lipid metabolism, and in hepatitis C virus replication (25,41). Krützfeldt et al. provide evidence to support miR-122 as a key regulator of the cholesterol biosynthetic pathway; in particular they observed that the expression of at least 11 genes involved in cholesterol biosynthesis was decreased between 1.4-fold and 2.3-fold in antagomir-122-treated mice, including hydroxy-3-methylglutaryl-CoA-reductase (Hmgcr), a rate-limiting enzyme of endogenous cholesterol biosynthesis. Observational and functional studies of miR-122 have highlighted this miRNA as a potential therapeutic target for the treatment of hypercholesterolemia and hepatitis C (25,41). Early antagonism of miR-122, using locked nucleic acid (LNA) modified DNA oligonucleotides (LNA-anti-miRs), resulted in effective silencing thus inhibiting HCV replication in HuH-7 cells harbouring the HCV-N replicon NNeo/C-5B (42). Silencing of miR-122 by systemic administration of high affinity LNA anti-miRs has resulted in dose dependent lowering of plasma cholesterol in mice and non-human primates (monkeys), after only three intravenous doses of 3 mg kg−1. Additionally, this was achieved without significant adverse sequelae or hepatic toxicity (41). These findings have unveiled the impending potential of miRNAs as novel therapeutic strategies. Indeed, a phase I safety and pharmacokinetic study of systemic miR-122 antagonism in humans using an LNA-based antisense molecule against miR-122 (SPC3649), led by Santaris Pharma, has been completed on 48 healthy volunteers and results are eagerly anticipated (43).
Novel biomarkers of the metabolic syndrome
Current challenges in the management of obesity and its related disorders include a search for unique biomarkers that are reflective or predictive of metabolic health and disease. Metabolic profiling has long been used to facilitate detection of disease states; indeed some of the first attempts to determine biomarkers of disease by global metabolic profiling were applied to the study of inborn errors of metabolism, where the relationship between disease state, genetics and the metabolic biomarker is easily understood. However, in many other metabolic diseases, the relationship between disease, genetics and metabolic state is complex and not readily understood. MiRNAs have been heralded as potential novel biomarkers for many pathological states, consequent to their tissue specific expression and association with clinicopathologic variables (Heneghan HM, Miller N, Lowery AJ, Sweeney KJ, Kerin MJ. J Oncol 2009. – in press). Their recent discovery in the circulation has prompted further exploration of their potential use as novel minimally invasive biomarkers of disease (44,45). In a preliminary study investigating this concept, Chen et al. have characterized the serum miRNA profile of diabetic patients and found that it differed significantly compared with healthy controls (44). It is unknown how miRNAs make their way into the bloodstream; however, Slack et al. raised two hypotheses in a recent report relating to the potential use of circulating miRNAs as tumour markers (46); first that tissue miRNAs may be present in circulation as a result of cell death and lyses, or alternatively that tissue cells actively secrete miRNAs into their microenvironment, where they enter blood vessels, and thereby make their way into the circulation. Future studies in this emerging field of research will provide a better understanding of the mechanisms by which miRNAs are released into the circulation. Further investigations in this field are also warranted to explore the ability of metabolic miRNA profiling to provide non-invasive translational biomarkers to reflect the state and extent of metabolic flux.
The association between aberrant miRNA expression and abnormalities in glucose homeostasis, adipogenesis and obesity, and functional analysis of specific miRNAs illustrates the feasibility of using these molecules as targets for therapeutic intervention (47). Antagomirs (cholesterol conjugated anti-sense oligonucleotides) are pioneering targets for miRNAs silencing, as evidenced by hepatic miR-122 blockade in vivo(41). These preliminary data have prompted early clinical trials. Conversely, where miRNA expression is known to be under-expressed (e.g. miR-103 in obese adipose tissue), induction of miRNA expression using viral or liposomal delivery of tissue-specific miRNAs to affected tissue could potentially result in restoration of catabolic activity to the tissue, although this concept of ‘miRNA replacement therapy’ has yet to be extrapolated in this setting. Further studies are necessary to examine the efficacy and safety of these novel therapeutic approaches, and to explore the potential for circulating miRNAs to aid in the management of patients with obesity and the metabolic syndrome; however, evidence to date is encouraging (44).
The documented involvement of miRNAs in glucose and lipid metabolism has provided strong evidence in support of their role as key players in the regulation of complex metabolic pathways. Additionally, it indicates potential for novel therapeutic strategies in the management of obesity and the metabolic syndrome. Further dedicated, focused research in this field is imperative to ascertain the full potential of miRNAs as novel metabolic biomarkers and potent therapeutic agents against obesity.