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Meta-regulation: microRNA regulation of glucose and lipid metabolism
Review
Meta-regulation: microRNA regulation of glucose and lipid metabolism Francis C. Lynn Departments of Surgery and Cellular and Physiological Sciences, Child and Family Research Institute, University of British Columbia, 950 W 28th St., Vancouver, British Columbia, V5Z 4H4, Canada
Maintenance of homeostasis during environmental flux requires constant metabolic adjustment, achieved partly through the fine regulation of gene expression. MicroRNAs are key players in this regulatory milieu; they have been implicated in regulating gene expression within several metabolically active tissues including the endocrine pancreas, liver and adipose tissue. Recent studies, for example, implicate miR-375 in pancreatic islet cell viability and function, and removal or overexpression of miR-375 profoundly affects glucose metabolism. In the liver, miR-122 is important for normal lipid metabolism. In fact, misexpression of miRNAs can occur in some diseases, suggesting that restoring miRNA expression is a potential therapeutic approach for both metabolic syndrome and diabetes. miRNAs: an emerging player in the regulatory milieu Gene expression can be controlled at the transcriptional level by the activity of DNA-binding transcription factors or controlled post-transcriptionally by changes in RNA stability or localization, protein translation or biological half-life. Since the description of the regulatory mechanisms active in the Escherichia coli operon, there has been great interest across phyla in cis regulatory-driven transcriptional control of gene expression through trans-acting protein factors. More recently, a similar paradigm has emerged that operates at the RNA level whereby transacting small (22 nucleotides) RNAs, microRNAs (miRNAs), play predominantly inhibitory regulatory roles by binding to cis-elements in the 30 untranslated region (UTR) of message-encoding RNAs. Over the past 5 years, it has become increasingly clear that miRNAs are not only important for normal organismal development and physiology, but also in the pathologies of diabetes, cancer, heart disease and inflammation. The importance of this new regulatory system was recently recognized with the presentation of the 2008 Albert Lasker Basic Medical Research Award to Victor Ambros, Gary Ruvkun and David Baulcombe, who pioneered miRNA research (Box 1). Transcription, processing and biological action of miRNAs In humans, approximately 720 miRNA genes are scattered throughout the genome; 70% of these are located in regions void of protein-coding genes and 30% within introns of protein-coding transcripts. Some miRNAs are situated in Corresponding author: Lynn, F.C. ([email protected]).
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independent transcriptional units; however, a great number are generated from either transcripts containing clusters of miRNAs or the intronic sequences of protein coding genes. Most miRNA transcription is carried out from regulated gene promoters by RNA polymerase II [1,2]. Once transcribed, the imperfect complementary sequence of the miRNAs leads to formation of complex secondary structure within the primary miRNA transcripts (primiRNA; Figure 1a). After folding, and while still within the nucleus, the double stranded (ds)RNA regions of the miRNAs are recognized and cleaved from the long transcript to form pre-miRNAs [3,4]. This cleavage step is catalyzed by the RNAse III-containing ‘‘microprocessor’’ enzyme complex, which is composed of RNASEN (DROSHA) and DGCR8 [4–6]. Following cleavage, the hairpin-loop pre-miRNAs are exported from the nucleus by exportin 5 in an energy-dependent process [7]. Once in the cytoplasm, the pre-miRNAs interact with a second double-strand specific RNAse III-containing enzyme complex, comprised of DICER 1 among other proteins. DICER 1 recognizes the dsRNA and cleaves approximately two helical turns or 22 bp from the non-loop end of the premiRNA [8], leaving a dsRNA that is then unwound by and incorporated into the RNA-induced silencing complex (RISC) [1,9,10]. The thermodynamic properties of the post-DICER1 dsRNA determine which strand will become the mature miRNA and which will be degraded [11,12]. Interestingly, both strands of a large number of pre-miRNAs can associate with RISC and carry out gene silencing. Once associated with the RISC, miRNAs are able to carry out the silencing of their repertoire of target genes. This occurs by one of three mechanisms that are not mutually exclusive: target cleavage, repression of target translation and message degradation in cytoplasmic Pbodies. When miRNA–target complementarity is high, pairing between the two results in message cleavage by RISC [10]. If miRNA–target complementarity is low, translational repression is often observed [2]. Often the latter effect relies on binding with near perfect complementarity of the miRNA ‘‘seed’’ region (nucleotides 2–8) to the 30 UTR of target mRNAs; these binding characteristics allow computational prediction of miRNA–target interactions [13]. However, accessibility of the target sequence in a complexly folded 30 UTR, binding with less than perfect ‘‘seed’’ complementarity and/or unanticipated protein–UTR interactions decrease the fidelity of these predictions and make experimental validation necessary [14]. Once the miRNA30 UTR interaction is established, target gene translational
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Review Box 1. The discovery of miRNAs in metazoans. The archetypical miRNA–target pair was defined in Caenorhabditis elegans in a series of papers published during the 1990s [60–63]. Several heterochronic mutants in worms had been described, including the lin-4, lin-14 and lin-28 mutants, which all affected the larval L1–L2 developmental transition [64,65]. Studies of the lin-14 gene by Ambros and Horvitz demonstrated that the retarded mutants of lin-14 were gain-of-function alleles and the precocious mutants of lin-14 were loss-of-function mutations [65,66]. Furthermore, these studies demonstrated that these two mutant classes manifested their respective phenotypes at distinct times during development [66]. At the same time, Ruvkin and Horvitz were undertaking studies aimed at understanding which portions of lin-14 were responsible for the lin-14 gain-of-function mutations. These studies demonstrated that all of the retarded mutants had deletions or rearrangements in the 30 UTR of the lin-14 gene [67,68]. Over the next few years, Ruvkin and Ambros demonstrated that lin-14 was downregulated at the protein level during development and hypothesized that lin-4 negatively regulated both lin-14 and lin-28 [69,70]. This led both groups to query both the molecular identity of lin-4 and question whether it was directly regulating lin-14. In backto-back studies, the Ambros and Ruvkin labs demonstrated that the active region of lin-4 could be traced to a non-protein coding region of 693 base pairs, which was processed into 21 and 61 nucleotide major and minor RNA fragments respectively, and that the 30 UTR of lin-14 conferred stage-specific post-transcriptional regulation on its expression. This resulted in stable expression of lin-14 at all stages of development and in the adult except at the L1–L2 transition, when lin-4 expression was elevated [60,63]. Both groups also noted that the small RNAs produced from the lin-4 locus were complimentary to several regions in the lin-14 30 UTR and concluded that interactions between the small RNAs and the lin-14 mRNA were responsible for lin-14 post-transcriptional regulation and downregulation at the L1-L2 transition [60,63]. Thus, the first miRNA– target interaction was defined. The importance of this discovery was not clear until cloning projects demonstrated that miRNAs were conserved and transcribed from genomes of all organisms, from plants to humans [71,72].
repression is thought to occur through a poorly defined and somewhat controversial mechanism that includes steric inhibition, sequestration of the message away from translationally competent ribosomes and/or message degradation [10]. Pancreatic islet miRNAs regulate glucose metabolism During embryonic development, the pancreatic primordia bud from the posterior foregut endoderm and the endocrine, exocrine and ductal structures, and subsequently differentiate through a complex branching morphogenic process [15]. Disruption of Dicer1 and subsequent loss of miRNAs early in pancreatic organogenesis leads to defects in formation of the pancreatic exocrine and endocrine systems [16]. Endocrine differentiation is affected by Dicer1 loss early in organ development, before activation of the pro-endocrine transcription factor neurogenin 3, and probably results from increased Notch signaling in progenitor cells [16]. Widespread expression of many miRNAs has been observed following endocrine specification in both mouse and human; however, the developmental relevance of many of these miRNAs is presently unclear [17–20]. One notable exception was the elegant demonstration by Kloosterman et al. that miRNA-375 is necessary for normal bcell clustering and islet formation in the zebrafish, a phenotype they attributed to loss of b-cell identity in miRNA-375 knockdown embryos [21].
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The first studies that implicated miRNAs in the control of vertebrate energy metabolism were carried out using pancreatic islets and cell lines [22,23]. In a seminal paper, Poy et al. cloned 11 novel miRNAs from pancreatic endocrine cell lines [24]. One of these, miRNA-375, was able to negatively regulate glucose-stimulated insulin secretion with no effect on either ATP production or intracellular calcium levels. These experiments suggested that miRNA375 was acting at a late step of insulin exocytosis, and capacitance measurements demonstrated that vesicular fusion was aberrant under conditions of miRNA-375 overexpression [24]. Computational methods predicted that Mtpn (myotrophin; Figure 2), a gene involved in actin depolymerization and potentially vesicular fusion, was a miRNA-375 target, and this prediction was subsequently experimentally validated [24]. In addition to regulating the trafficking of vesicles towards the plasma membrane, it is likely that miRNAs also regulate insulin secretion at other points in the secretory pathway. Granuphilin (SYTL4), a synaptotagmin-like protein that has been demonstrated to be important for insulin vesicle docking, was shown to be regulated indirectly through miRNA-9 regulation of Onecut2 expression [25,26]. Another study from the same group demonstrated that miRNA-124a2, miRNA-9 and miRNA-96 could all regulate the expression of proteins involved in insulin vesicle docking and exocytosis in MIN6 B1 pancreatic b-cells [27]. In this study, miRNA-96 and miRNA-9 negatively regulated insulin secretion through upregulation of granuphilin. miRNA-124a2 increased basal insulin secretion by regulating multiple exocytotic genes; however, only Rab27a was found to be a direct target of miRNA124a2 [27]. In a separate study, miRNA-124a2 was shown to directly regulate Foxa2 expression and thereby control the levels of pancreatic and duodenal homeobox 1 (Pdx1), the ATP-sensitive potassium channel (Kcnj11), the sulfonyl urea receptor (Abcc8) and insulin mRNA without marked effects on insulin secretion [28]. Future studies should be aimed at understanding whether the indirect effects of miRNA-124a2 on b-cell exocytotic proteins are mediated through its targeting of Foxa2. The dysregulation of glucose-stimulated insulin secretion is a hallmark of diabetes. Several miRNAs have been found to be differentially expressed in high glucose conditions including miRNA-124a2, -34a, -107, -30d, -9, -296, -484, -690 and -375 [29–31]. High glucose increased miRNA-30d expression, resulting in increased insulin gene expression with no significant effect on insulin secretion [31]. It is unclear if this is a transcriptional or post-transcriptional effect. However, neither Pdx1 nor NeuroD1, two transcription factors important for insulin expression, were induced when miRNA-30d was overexpressed, ruling out these specific insulin gene regulators as mediators of this effect. High glucose decreased expression of miRNA375 in both rat INS-1E cells and in isolated rat islets (Figure 2e) [29]. This decrease in miRNA-375 expression was correlated with increases in both 30 -phosphoinositodedependent protein kinase 1 (Pdpk1) and insulin gene expression, and it was subsequently demonstrated that Pdpk1 is a direct target of miRNA-375 [29]. Knockdown of miRNA-375 led to increased Pdpk1 and insulin expression, 453
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Figure 1. The biogenesis and function of miRNAs. (a) miRNAs are transcribed by RNA polymerase II as long primary trancripts, pri-miRNA. Upon transcription, the miRNAcontaining region of pre-miRNA forms a hairpin-loop, which is recognised by (b) the ‘‘microprocessor’’ complex composed of DGCR8 (blue oval) and Drosha (purple star). Drosha then cleaves the pre-miRNA hairpin-loop from the primary transcript, and (c) pre-miRNAs are exported from the nucleus by exportin 5. (d) Once in the cytoplasm, DICER 1 binds to pre-miRNA and the loop is removed, leaving a small double-stranded RNA. (e) The DICER 1-bound dsRNA then rapidly associates with the RNA-induced silencing complex (RISC), which is composed of the TAR RNA binding protein 2 (TRBP), an argonaute protein (AGO) and GW182, among others. Interaction with RISC leads to unwinding of the double stranded small RNA, and the miRNA strand (red strand) remains bound to AGO. The RISC bound miRNA is then able to carry out gene silencing. (f) RNA cleavage occurs in targets that display a fully complementary miRNA binding site and (g) translational repression occurs with partial complementarity between miRNA and its target.
the latter presumably through increased PDPK1 signaling (Figure 2c) [29]. These two studies highlight the importance of miRNAs in b-cell glucose responsiveness. Undoubtedly, future studies will focus on how other miRNAs regulate b-cell glucose sensing and how dysregulation of miRNA expression contributes to the loss of glucose competence and type 2 diabetes. Obesity leads to compensatory expansion of b-cell mass through increased b-cell replication. In type 2 diabetes, an increase in b-cell apoptosis concurrent with increased insulin demand leads to hyperglycemia. There has been substantial interest in understanding whether miRNAs regulate b-cell mass and whether they could be used as therapeutic agents for diabetes treatment. Lovis et al. [30] demonstrated that miRNA-34a is induced in a tumor protein p53-dependent manner in MIN6 B1 mouse b-cells by free fatty acids and in the db/db diabetic mouse model, and that miRNA-34a significantly increased apoptosis under normal conditions by reducing Bcl2 expression in MIN6 B1 cells. Thus, it is possible that therapeutic suppression of miRNA-34a could prevent b-cell loss during the development of diabetes [30]. In a recent study, Poy et al. reported the generation and phenotyping of the miRNA-375 knockout mouse (Figure 2d,f; Table 1) [32]. These animals display marked hyperglycemia that resulted from increased hepatic glucose output caused by increased a-cell mass and hyperglucago454
nemia, without an apparent increase in glucagon secretion per cell [32]. As in the earlier study [24], b-cell insulin secretory capacity was enhanced in these animals, but this increase was offset by a dramatic decrease in b-cell mass, attributed to increased expression of genes that negatively regulate b-cell replication (Figure 2d; Table 1) [32]. Because miRNA-375 expression was elevated in the ob/ob mouse [32], the miRNA-375 knockout was crossed into the ob/ob background to determine if miRNA-375 upregulation is important for the b-cell compensation observed in obesity. As early as 4 weeks of age, the double miRNA-375/leptin null animals (DKO) displayed significant hyperglycemia compared with either single knockout strain; by 10 weeks, the DKO showed significant reductions in b-cell replication and plasma insulin concomitant with increased hepatic glucose output [32]. It is unclear why a-cell replication increases whereas b-cell replication decreases in the miRNA-375 knockout animal (Figure 2f). It is hoped that future studies will address which genes are necessary for these effects in each cell type and clarify whether miRNA-375 loss results in a subtle but physiologically relevant increase in b-cell apoptosis. Liver miRNAs and lipid metabolism Once secreted from the islet, insulin and glucagon travel to the liver through the portal circulation where they control hepatic glucose and lipid metabolism. Liver insulin
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Figure 2. The role of miRNA-375 in pancreatic islet function. In the pancreatic b-cell (yellow), (a) miRNA-375 interacts with the myotrophin (Mtpn) mRNA leading to both down-regulation of mRNA expression and a decreased translation (red octagon). Knockdown of myotrophin results in suppression of insulin secretion possibly through the regulation of the actin cytoskeleton (F-Actin). (b) Insulin might regulate its own expression through binding to cell surface insulin receptors (INSR) and stimulating a phosphorylation-dependent signaling cascade that results in the phosphorylation of 3-phosphoinositide dependent protein kinase-1 (PDPK1). PDPK1 can then phosphorylate protein kinase B (AKT) and AKT, in turn, phosphorylates glycogen synthase kinase 3 beta (GSK3B), which phosphorylates the pancreatic and duodenal homeobox 1 (PDX1) transcription factor. Upon phosphorylation, pPDX1 translocates to the nucleus where it stimulates insulin transcription. (c) Pdpk1 is negatively regulated by miRNA-375, resulting in decreased insulin gene transcription. (d) Germline removal of miRNA-375 in mice results in the induction of several genes that are important for restraining cell replication; therefore, miRNA-375 is believed to normally stimulate expansion of b-cell mass through partial inhibition of these pathways. Glucose is important for b-cell replication, insulin gene transcription and insulin secretion. (e) Glucose represses expression of miRNA-375 through an undetermined mechanism thereby reversing all of these repressive effects (a–d). (f) The islet a-cell (blue) also expresses miRNA-375, and mice with null mutations in the miRNA-375 gene display markedly elevated plasma glucagon levels and increased a-cell proliferation. It is unclear which miRNA-375 target genes normally repress both a-cell replication and glucagon secretion. ?, unknown.
resistance plays an important role in the development of the metabolic syndrome. and dysregulation of miRNA expression potentially influences insulin resistance. Dicer1 deletion from the early postnatal liver resulted in profound hepatocyte apoptosis, steatosis and mild hypoglycemia [33]. The most highly expressed miRNA in the liver is miRNA122, which has been estimated to be expressed at up to 135,000 copies per human hepatocyte [34]. Several groups have assessed how miRNA-122 might affect hepatocyte biology by using antisense methods to suppress miRNA122 expression; all have shown that this miRNA is important for regulating liver lipid metabolism (Table 1) [35–38]. Mice treated with cholesterol-conjugated, 20 -O-methyl oligonucleotides antisense to miRNA-122 showed dramatic
reductions in peripheral expression of miRNA-122, with nearly complete loss in the liver [35,38]. This resulted in upregulation, at the message level, of a large number of genes that contained the miRNA-122 ‘‘seed’’ binding sequences in their 30 UTRs [38]. Knockdown of miRNA122 in mice decreased expression of several genes important for cholesterol biosynthesis, whereas adenoviral overexpression of miRNA-122 increased cholesterol biosynthesis. However, the effects of miRNA-122 on cholesterol biosynthesis are indirect, and it is unclear which direct targets of miRNA-122 mediate them [38]. A second complementary study found that miRNA-122 knockdown in mice decreased circulating cholesterol levels, liver cholesterol and fatty acid synthesis and increased liver fatty acid oxidation [37]. This 455
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Table 1. Target-miRNA pairs that have been experimentally validated as important for glucose and lipid metabolism Tissue/cell type Pancreatic Islet MIN6 mouse b-cells INS IE rat b-cells miR-375 knockout mouse islets
INS IE rat b-cells MIN6 Bl mouse b-cells MIN6 mouse b-cells MIN6 Bl mouse b-cells Liver primary mouse hepatocytes primary mouse hepatocytes primary mouse hepatocytes
miRNA
Biological Result of miRNA Expression
Putative Target Gene
Reference
miR-375
Suppression of insulin secretion Suppression of insulin transcription Cell growth, possibly through increased cell cycling Cell growth, possibly via reduced caspase-2 activity Cell growth, possibly via maintence of genomic stability Cell growth, via reduction in apoptosis Cell growth, via unknown mechanism Cell growth, possibly via increased Gi/Gq signalling Cell growth, possibly via reduction of p53 activity Cell growth, possibly via reduction in apoptosis Cell growth, possibly via increased cell cycling Cell growth, unknown mechanism Suppression of insulin secretion Dysregulation of insulin secretion Suppression of insulin transcription, possible increase in secretion Suppression of insulin secretion Sensitization to apoptosis
Mtpn Pdkl Cavl Id3 Smarca2 Aifml Rasdl Rgsl6 Eeflel Clqbp HuD Cadml Onecut2 Rab27A Foxa2 Vamp2 Bcl2
[24] [29] [32] [32] [32] [32] [32] [32] [32] [32] [32] [32] [26] [27] [28] [30] [30]
miR-122
Unknown, possibly reduced intracellular amino acid concentration Cholesterol synthesis, possibly reduced glycolysis Cholesterol synthesis, possibly reduced glycogenesis Synthesis, possibly increased cell cycling Cholesterol synthesis, possibly reduced RNAi Cholesterol synthesis, unknown role Cholesterol synthesis, unknown role Cholesterol synthesis, unknown role Cholesterol synthesis, unknown role Dysregulation of cholesterol synthesis and lipogenesis Dysregulation of cholesterol synthesis and lipogenesis Dysregulation cholesterol synthesis and lipogenesis Dysregulation cholesterol synthesis and lipogenesis
Slc7al Aldoa Gysl Ccngl P4hal Ndrg3 Tmed3 Hfe2 Slc35a4 SREBP2 HMGCR SREBPlc FAS
[34,37] [35,37,38] [37] [37] [37] [38] [38] [38] [38] [39] [39] [39] [39]
miR-278
Insulin sensitivity in Drosophila
expanded
[46]
miR-9 miR-124a miR-34a
primary mouse hepatocytes
HepG2 human hepatoma
Adipose
study also demonstrated that miRNA-122 inhibition reduced both hepatic cholesterol accumulation and liver steatosis during the development of diet-induced obesity in mice. Finally, these authors demonstrated an increase in phosphorylated AMP-activated protein kinase (AMPK) in livers of these mice, but failed to clarify if miRNA-122 was directly regulating AMPK signaling [37]. A third study in primates found that inhibition of miRNA-122 with antisense molecules reduced plasma cholesterol levels without any apparent liver toxicity [36], suggesting miRNA inhibition as a feasible therapeutic approach in humans. miRNA-122 has been demonstrated to be important for liver fat metabolism, but there is also some evidence that dysregulation of miRNA expression might underlie some disease pathologies [39]. Nonalcoholic fatty liver disease (NAFLD) has been demonstrated to be present in a large percentage of individuals with metabolic syndrome, and might be partly responsible for the development of liver insulin resistance [40]. In one study, 46 miRNAs were differentially expressed in humans with NAFLD [39]. miRNA-122 was in the group of genes that was downregulated in NAFLD, and this was correlated with increased expression of lipogenic genes in the human livers. Knockdown of miRNA-122 in HepG2 cells recapitulated the lipogenic gene expression profile observed in individuals with NAFLD [39]. In this case, it seems likely that miRNA-122 downregulation is a compensatory mechanism that counters increasing liver lipid levels, rather 456
than a causative agent in the development of NAFLD. Future studies should clarify this apparent inconsistency. In any case, the future use of miRNA-122 mimetics or antagonists could rebalance the defects observed in the liver during metabolic syndrome [41]. Adipose tissue miRNAs and energy metabolism Fatty acids travel in lipoprotein particles from the liver to the adipose tissue where they are stored in lipid droplets. Knockdown of either DICER1 or RNASEN prevents in vitro differentiation of human multipotent stromal cells to adipocytes, implying that miRNAs might be important for adipogenesis [42]. The first indication that miRNAs might be important in adipose cell biology came from the observation that miRNA-143 is one of several miRNAs that are upregulated during differentiation of human pre-adipocytes [43]. Inhibition of miRNA-143 action by antisense oligonucleotides led to a substantial inhibition of differentiation with decreases in triglyceride accumulation, and reductions in expression of the adipocyte-specific genes fatty acid binding protein 4 (FABP4), glucose transporter 4 (SLC2A4), peroxisome proliferator activated receptor gamma (PPARG) and hormone sensitive lipase (LIPE) [43]. Mitogen-activated protein kinase 7 (MAPK7) was shown to be upregulated by knockdown of miRNA-143 in these experiments but its role in adipogenesis is presently unclear [43]. Xie et al. found that both miRNA-143 and -103 were upregulated during adipogenesis in mouse 3T3-L1
Review preadipocytes [44]. Furthermore, overexpressing these two miRNAs during adipogenesis resulted in significantly increased expression of several adipocyte-specific genes. These two miRNAs are markedly downregulated, possibly through an inflammatory pathway in the development of obesity in both ob/ob mice and mice fed a high-fat diet [44]. The first report implicating miRNA involvement in adipose-regulated energy metabolism was carried out using Drosophila. miRNA-14 null adult flies have substantially increased lipid droplet accumulation in adipose tissue. In addition, whole body increases in both di- and triacylglycerol levels were observed, and these were correlated with increased apoptosis in several tissues in the mutant flies [45]. A second study in flies demonstrated that miRNA-278 mutants had reduced whole body fat levels [46], yet these mutant flies also displayed elevated insulin and sugar levels, primarily in the form of trehalose, which is generated from mobilisation of glycogen reserves in the Drosophila adipose tissue analog. The observation of elevated sugars and loss of body fat in the face of elevated insulin levels suggests that the fat body in the miRNA-278 mutant is resistant to insulin [46]. This hypothesis was confirmed by the observations that expression levels of the insulin target genes 4E-BP and InR were inappropriately high in the mutant fly fat bodies. Overexpression of the putative miRNA-278 target gene expanded (ex), which can act as a tumor suppressor, was able to phenocopy the miRNA-278 knockout, suggesting that ex is a likely biological target of miRNA-278 in Drosophila (Table 1) [46]. Although neither miRNA-278 nor miRNA-14 is expressed in higher organisms, similar phenotypes are observed upon loss of insulin signaling in adipose tissue in mammals. For example, mice that have the insulin receptor gene selectively deleted from adipose tissue are lean and have a reduction in fat pad mass, whereas mice that have the insulin receptor gene removed from muscle show increased adipose tissue stores [47,48]. One study has directly addressed the role of miRNA-29 in the development of insulin resistance in mammals. This study demonstrated that miRNA-29a, b and c were all upregulated in adipose tissue of diabetic rats, and that this upregulation could be induced by exposing 3T3-L1 mouse adipocytes to insulin and glucose [49]. Overexpressing miRNA-29 in the same adipocytes reduced insulin-stimulated glucose uptake, a reduction that was attributed to disrupted insulin signaling through a reduction in insulin-stimulated AKT1 phosphorylation. However, it remains unclear which miRNA-29 target genes mediate these effect in adipocytes [49]. Adipocytes can be functionally characterized into two groups: energy-storing white adipocytes and energyexpending brown adipocytes. Recently, it was demonstrated that brown adipocytes and muscle share a common precursor, and that the zinc finger transcription factor PRDM16 regulates the differentiation of brown fat at the expense of muscle [50]. In line with their common embryological origins, brown adipose tissue and muscle share similar patterns of miRNA expression, with low levels of miRNA-143 and higher levels of miRNA-1, -133a and -206 [51]. The physiological significance of this expression profile, however, has not yet been addressed.
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Future studies might be targeted at understanding how additional miRNAs regulate adipocyte insulin sensitivity, adipokine and adipose-derived hormone secretion, and lipolysis both normally and in disease. Muscle and brain miRNAs and metabolic control Muscle is a major site of glucose disposal and metabolism. Although there is substantial evidence demonstrating a role for miRNAs in muscle growth and development [52], little is known about miRNA regulation of muscle glucose metabolism. Dicer1 removal from cardiac progenitors, from adult hearts and from developing muscle all lead to tissue loss and death [53–55]. The miRNA-1/206 and the miRNA133a/133b families appear to be most important for muscle development and growth. These miRNAs are all regulated by one or more of the myogenic transcription factors, and are important for specification and function of both cardiac and skeletal muscle [52]. It has been demonstrated that some miRNAs are differentially expressed in the skeletal muscle of diabetic GK rats; however, the physiological relevance of this observation is unknown [49]. It is hoped that future studies will focus on understanding whether miRNAs can regulate insulin signaling in muscle and whether ‘‘myomiR’’ levels are dysregulated during metabolic disturbances and pathologies. Circulating nutrients and hormones not only affect peripheral tissues but also the feeding and satiety centers in the hypothalamus; in turn, afferent signals from the brain regulate pancreatic islet, liver and adipose physiology [56]. Thus, the brain should be considered part of the system responsible for the regulation of carbohydrate and fat metabolism. A plethora of miRNAs is expressed in the nervous system, and removal of Dicer1 in specific brain regions can lead to both behavioral defects and neurodegeneration [57–59]. The role of miRNAs in the feeding and satiety centers has not been addressed; however, as in all the other metabolically active tissues, they undoubtedly play a role in these neurons as well. Concluding remarks It is now clear that gene regulatory mechanisms governed by miRNAs are important for the development and maintenance of metabolically active tissues. Furthermore, alteration in miRNA expression in several of these tissues can result in impaired glucose and lipid homeostasis through a wide range of mechanisms ranging from cell cycle control to exocytotic regulation. Unfortunately, the regulatory power that miRNAs exert through their large repertoire of target genes is the Achilles heel of miRNA research, as it is difficult to dissect out the specific factors that are important for any one phenotype. In the future, it will be important to develop experimental methods that enable a more integrative approach to miRNA biology including hierarchical assignment of target relevance, regulation of tissue-specific miRNA expression and processing during disease, development of methods for the facile generation of multiallelic knockouts to study miRNA families, and novel delivery methods for tissuespecific therapeutics. These tiny regulators are clearly important for normal biology and in disease etiology, and their regulation of regulators of glucose and lipid 457
Review metabolism or ‘‘meta-regulation’’ clearly warrants further research. Acknowledgements I am indebted to Michael German, Matthias Hebrok and Michael McManus for their help, support and advice. Postdoctoral fellowship awards 3-2004-276 and 10-2007-86 from Juvenile Diabetes Research Foundation allowed me to carry out studies in this field and for this I am grateful. This is a burgeoning field and as such there has been a huge increase in the number of publications over the past 5 years. This large number of exciting publications has made this article possible; however, the length of this review precludes citation of original manuscripts and I apologise for this.
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Trends in Endocrinology and Metabolism Vol.20 No.9 27 Lovis, P. et al. (2008) Regulation of the expression of components of the exocytotic machinery of insulin-secreting cells by microRNAs. Biol. Chem. 389, 305–312 28 Baroukh, N. et al. (2007) MicroRNA-124a regulates Foxa2 expression and intracellular signaling in pancreatic beta-cell lines. J. Biol. Chem. 282, 19575–19588 29 El Ouaamari, A. et al. (2008) miR-375 targets 3’-phosphoinositidedependent protein kinase-1 and regulates glucose-induced biological responses in pancreatic beta-cells. Diabetes 57, 2708–2717 30 Lovis, P. et al. (2008) Alterations in microRNA expression contribute to fatty acid-induced pancreatic beta-cell dysfunction. Diabetes 57, 2728– 2736 31 Tang, X. et al. (2009) Identification of glucose-regulated miRNAs from pancreatic {beta} cells reveals a role for miR-30d in insulin transcription. RNA 15, 287–293 32 Poy, M.N. et al. (2009) miR-375 maintains normal pancreatic {alphaand {beta-cell mass. Proc. Natl. Acad. Sci. U. S. A. 106, 5813–5818 33 Sekine, S. et al. (2009) Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis. Gastroenterology 136, 2304–2315 34 Chang, J. et al. (2004) miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA Biol. 1, 106–113 35 Davis, S. et al. (2009) Potent inhibition of microRNA in vivo without degradation. Nucleic Acids Research 37, 70–77 36 Elme´n, J. et al. (2008) LNA-mediated microRNA silencing in nonhuman primates. Nature 452, 896–899 37 Esau, C. et al. (2006) miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab. 3, 87–98 38 Kru¨tzfeldt, J. et al. (2005) Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438, 685–689 39 Cheung, O. et al. (2008) Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression. Hepatology 48, 1810–1820 40 Tilg, H. and Moschen, A.R. (2008) Insulin resistance, inflammation, and non-alcoholic fatty liver disease. Trends Endocrinol. Metab. 19, 371–379 41 Bonetta, L. (2009) RNA-based therapeutics: ready for delivery? Cell 136, 581–584 42 Oskowitz, A.Z. et al. (2008) Human multipotent stromal cells from bone marrow and microRNA: regulation of differentiation and leukemia inhibitory factor expression. Proc. Natl. Acad. Sci. U. S. A. 105, 18372–18377 43 Esau, C. et al. (2004) MicroRNA-143 regulates adipocyte differentiation. J. Biol. Chem. 279, 52361–52365 44 Xie, H. et al. (2009) MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity. Diabetes 58, 1050–1057 45 Xu, P. et al. (2003) The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism. Curr. Biol. 13, 790– 795 46 Teleman, A.A. et al. (2006) Drosophila lacking microRNA miR-278 are defective in energy homeostasis. Genes Dev. 20, 417–422 47 Blu¨her, M. et al. (2002) Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance. Dev. Cell 3, 25–38 48 Bru¨ning, J.C. et al. (1998) A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol. Cell 2, 559–569 49 He, A. et al. (2007) Overexpression of micro ribonucleic acid 29, highly up-regulated in diabetic rats, leads to insulin resistance in 3T3-L1 adipocytes. Mol.Endocrinol. 21, 2785–2794 50 Seale, P. et al. (2008) PRDM16 controls a brown fat/skeletal muscle switch. Nature 454, 961–967 51 Walden, T.B. et al. (2009) Distinct expression of muscle-specific microRNAs (myomirs) in brown adipocytes. J. Cell. Physiol. 218, 444–449 52 Williams, A.H. et al. (2009) MicroRNA control of muscle development and disease. Curr. Opin. Cell. Biol. 21, 461–469 53 Chen, J.F. et al. (2008) Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure. Proc. Natl. Acad. Sci. U. S. A. 05, 2111–2116 54 O’Rourke, J.R. et al. (2007) Essential role for Dicer during skeletal muscle development. Dev. Biol. 311, 359–368
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63 Wightman, B. et al. (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75, 855–862 64 Chalfie, M. et al. (1981) Mutations that lead to reiterations in the cell lineages of C. elegans. Cell 24, 59–69 65 Ambros, V. and Horvitz, H.R. (1984) Heterochronic mutants of the nematode Caenorhabditis elegans. Science 226, 409–416 66 Ambros, V. and Horvitz, H.R. (1987) The lin-14 locus of Caenorhabditis elegans controls the time of expression of specific postembryonic developmental events. Genes Dev. 1, 398–414 67 Ruvkun, G. et al. (1989) Molecular genetics of the Caenorhabditis elegans heterochronic gene lin-14. Genetics 121, 501–516 68 Wightman, B. et al. (1991) Negative regulatory sequences in the lin-14 3’-untranslated region are necessary to generate a temporal switch during Caenorhabditis elegans development. Genes Dev. 5, 1813–1824 69 Ambros, V. (1989) A hierarchy of regulatory genes controls a larva-toadult developmental switch in C. elegans. Cell 57, 49–57 70 Ruvkun, G. and Giusto, J. (1989) The Caenorhabditis elegans heterochronic gene lin-14 encodes a nuclear protein that forms a temporal developmental switch. Nature 338, 313–319 71 Lagos-Quintana, M. et al. (2001) Identification of novel genes coding for small expressed RNAs. Science 294, 853–858 72 Pasquinelli, A.E. et al. (2000) Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 408, 86–89
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Meta-regulation: microRNA regulation of glucose and lipid metabolism Francis C. Lynn Departments of Surgery and Cellular and Physiological Sciences, Child and Family Research Institute, University of British Columbia, 950 W 28th St., Vancouver, British Columbia, V5Z 4H4, Canada
Maintenance of homeostasis during environmental flux requires constant metabolic adjustment, achieved partly through the fine regulation of gene expression. MicroRNAs are key players in this regulatory milieu; they have been implicated in regulating gene expression within several metabolically active tissues including the endocrine pancreas, liver and adipose tissue. Recent studies, for example, implicate miR-375 in pancreatic islet cell viability and function, and removal or overexpression of miR-375 profoundly affects glucose metabolism. In the liver, miR-122 is important for normal lipid metabolism. In fact, misexpression of miRNAs can occur in some diseases, suggesting that restoring miRNA expression is a potential therapeutic approach for both metabolic syndrome and diabetes. miRNAs: an emerging player in the regulatory milieu Gene expression can be controlled at the transcriptional level by the activity of DNA-binding transcription factors or controlled post-transcriptionally by changes in RNA stability or localization, protein translation or biological half-life. Since the description of the regulatory mechanisms active in the Escherichia coli operon, there has been great interest across phyla in cis regulatory-driven transcriptional control of gene expression through trans-acting protein factors. More recently, a similar paradigm has emerged that operates at the RNA level whereby transacting small (22 nucleotides) RNAs, microRNAs (miRNAs), play predominantly inhibitory regulatory roles by binding to cis-elements in the 30 untranslated region (UTR) of message-encoding RNAs. Over the past 5 years, it has become increasingly clear that miRNAs are not only important for normal organismal development and physiology, but also in the pathologies of diabetes, cancer, heart disease and inflammation. The importance of this new regulatory system was recently recognized with the presentation of the 2008 Albert Lasker Basic Medical Research Award to Victor Ambros, Gary Ruvkun and David Baulcombe, who pioneered miRNA research (Box 1). Transcription, processing and biological action of miRNAs In humans, approximately 720 miRNA genes are scattered throughout the genome; 70% of these are located in regions void of protein-coding genes and 30% within introns of protein-coding transcripts. Some miRNAs are situated in Corresponding author: Lynn, F.C. ([email protected]).
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independent transcriptional units; however, a great number are generated from either transcripts containing clusters of miRNAs or the intronic sequences of protein coding genes. Most miRNA transcription is carried out from regulated gene promoters by RNA polymerase II [1,2]. Once transcribed, the imperfect complementary sequence of the miRNAs leads to formation of complex secondary structure within the primary miRNA transcripts (primiRNA; Figure 1a). After folding, and while still within the nucleus, the double stranded (ds)RNA regions of the miRNAs are recognized and cleaved from the long transcript to form pre-miRNAs [3,4]. This cleavage step is catalyzed by the RNAse III-containing ‘‘microprocessor’’ enzyme complex, which is composed of RNASEN (DROSHA) and DGCR8 [4–6]. Following cleavage, the hairpin-loop pre-miRNAs are exported from the nucleus by exportin 5 in an energy-dependent process [7]. Once in the cytoplasm, the pre-miRNAs interact with a second double-strand specific RNAse III-containing enzyme complex, comprised of DICER 1 among other proteins. DICER 1 recognizes the dsRNA and cleaves approximately two helical turns or 22 bp from the non-loop end of the premiRNA [8], leaving a dsRNA that is then unwound by and incorporated into the RNA-induced silencing complex (RISC) [1,9,10]. The thermodynamic properties of the post-DICER1 dsRNA determine which strand will become the mature miRNA and which will be degraded [11,12]. Interestingly, both strands of a large number of pre-miRNAs can associate with RISC and carry out gene silencing. Once associated with the RISC, miRNAs are able to carry out the silencing of their repertoire of target genes. This occurs by one of three mechanisms that are not mutually exclusive: target cleavage, repression of target translation and message degradation in cytoplasmic Pbodies. When miRNA–target complementarity is high, pairing between the two results in message cleavage by RISC [10]. If miRNA–target complementarity is low, translational repression is often observed [2]. Often the latter effect relies on binding with near perfect complementarity of the miRNA ‘‘seed’’ region (nucleotides 2–8) to the 30 UTR of target mRNAs; these binding characteristics allow computational prediction of miRNA–target interactions [13]. However, accessibility of the target sequence in a complexly folded 30 UTR, binding with less than perfect ‘‘seed’’ complementarity and/or unanticipated protein–UTR interactions decrease the fidelity of these predictions and make experimental validation necessary [14]. Once the miRNA30 UTR interaction is established, target gene translational
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Review Box 1. The discovery of miRNAs in metazoans. The archetypical miRNA–target pair was defined in Caenorhabditis elegans in a series of papers published during the 1990s [60–63]. Several heterochronic mutants in worms had been described, including the lin-4, lin-14 and lin-28 mutants, which all affected the larval L1–L2 developmental transition [64,65]. Studies of the lin-14 gene by Ambros and Horvitz demonstrated that the retarded mutants of lin-14 were gain-of-function alleles and the precocious mutants of lin-14 were loss-of-function mutations [65,66]. Furthermore, these studies demonstrated that these two mutant classes manifested their respective phenotypes at distinct times during development [66]. At the same time, Ruvkin and Horvitz were undertaking studies aimed at understanding which portions of lin-14 were responsible for the lin-14 gain-of-function mutations. These studies demonstrated that all of the retarded mutants had deletions or rearrangements in the 30 UTR of the lin-14 gene [67,68]. Over the next few years, Ruvkin and Ambros demonstrated that lin-14 was downregulated at the protein level during development and hypothesized that lin-4 negatively regulated both lin-14 and lin-28 [69,70]. This led both groups to query both the molecular identity of lin-4 and question whether it was directly regulating lin-14. In backto-back studies, the Ambros and Ruvkin labs demonstrated that the active region of lin-4 could be traced to a non-protein coding region of 693 base pairs, which was processed into 21 and 61 nucleotide major and minor RNA fragments respectively, and that the 30 UTR of lin-14 conferred stage-specific post-transcriptional regulation on its expression. This resulted in stable expression of lin-14 at all stages of development and in the adult except at the L1–L2 transition, when lin-4 expression was elevated [60,63]. Both groups also noted that the small RNAs produced from the lin-4 locus were complimentary to several regions in the lin-14 30 UTR and concluded that interactions between the small RNAs and the lin-14 mRNA were responsible for lin-14 post-transcriptional regulation and downregulation at the L1-L2 transition [60,63]. Thus, the first miRNA– target interaction was defined. The importance of this discovery was not clear until cloning projects demonstrated that miRNAs were conserved and transcribed from genomes of all organisms, from plants to humans [71,72].
repression is thought to occur through a poorly defined and somewhat controversial mechanism that includes steric inhibition, sequestration of the message away from translationally competent ribosomes and/or message degradation [10]. Pancreatic islet miRNAs regulate glucose metabolism During embryonic development, the pancreatic primordia bud from the posterior foregut endoderm and the endocrine, exocrine and ductal structures, and subsequently differentiate through a complex branching morphogenic process [15]. Disruption of Dicer1 and subsequent loss of miRNAs early in pancreatic organogenesis leads to defects in formation of the pancreatic exocrine and endocrine systems [16]. Endocrine differentiation is affected by Dicer1 loss early in organ development, before activation of the pro-endocrine transcription factor neurogenin 3, and probably results from increased Notch signaling in progenitor cells [16]. Widespread expression of many miRNAs has been observed following endocrine specification in both mouse and human; however, the developmental relevance of many of these miRNAs is presently unclear [17–20]. One notable exception was the elegant demonstration by Kloosterman et al. that miRNA-375 is necessary for normal bcell clustering and islet formation in the zebrafish, a phenotype they attributed to loss of b-cell identity in miRNA-375 knockdown embryos [21].
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The first studies that implicated miRNAs in the control of vertebrate energy metabolism were carried out using pancreatic islets and cell lines [22,23]. In a seminal paper, Poy et al. cloned 11 novel miRNAs from pancreatic endocrine cell lines [24]. One of these, miRNA-375, was able to negatively regulate glucose-stimulated insulin secretion with no effect on either ATP production or intracellular calcium levels. These experiments suggested that miRNA375 was acting at a late step of insulin exocytosis, and capacitance measurements demonstrated that vesicular fusion was aberrant under conditions of miRNA-375 overexpression [24]. Computational methods predicted that Mtpn (myotrophin; Figure 2), a gene involved in actin depolymerization and potentially vesicular fusion, was a miRNA-375 target, and this prediction was subsequently experimentally validated [24]. In addition to regulating the trafficking of vesicles towards the plasma membrane, it is likely that miRNAs also regulate insulin secretion at other points in the secretory pathway. Granuphilin (SYTL4), a synaptotagmin-like protein that has been demonstrated to be important for insulin vesicle docking, was shown to be regulated indirectly through miRNA-9 regulation of Onecut2 expression [25,26]. Another study from the same group demonstrated that miRNA-124a2, miRNA-9 and miRNA-96 could all regulate the expression of proteins involved in insulin vesicle docking and exocytosis in MIN6 B1 pancreatic b-cells [27]. In this study, miRNA-96 and miRNA-9 negatively regulated insulin secretion through upregulation of granuphilin. miRNA-124a2 increased basal insulin secretion by regulating multiple exocytotic genes; however, only Rab27a was found to be a direct target of miRNA124a2 [27]. In a separate study, miRNA-124a2 was shown to directly regulate Foxa2 expression and thereby control the levels of pancreatic and duodenal homeobox 1 (Pdx1), the ATP-sensitive potassium channel (Kcnj11), the sulfonyl urea receptor (Abcc8) and insulin mRNA without marked effects on insulin secretion [28]. Future studies should be aimed at understanding whether the indirect effects of miRNA-124a2 on b-cell exocytotic proteins are mediated through its targeting of Foxa2. The dysregulation of glucose-stimulated insulin secretion is a hallmark of diabetes. Several miRNAs have been found to be differentially expressed in high glucose conditions including miRNA-124a2, -34a, -107, -30d, -9, -296, -484, -690 and -375 [29–31]. High glucose increased miRNA-30d expression, resulting in increased insulin gene expression with no significant effect on insulin secretion [31]. It is unclear if this is a transcriptional or post-transcriptional effect. However, neither Pdx1 nor NeuroD1, two transcription factors important for insulin expression, were induced when miRNA-30d was overexpressed, ruling out these specific insulin gene regulators as mediators of this effect. High glucose decreased expression of miRNA375 in both rat INS-1E cells and in isolated rat islets (Figure 2e) [29]. This decrease in miRNA-375 expression was correlated with increases in both 30 -phosphoinositodedependent protein kinase 1 (Pdpk1) and insulin gene expression, and it was subsequently demonstrated that Pdpk1 is a direct target of miRNA-375 [29]. Knockdown of miRNA-375 led to increased Pdpk1 and insulin expression, 453
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Figure 1. The biogenesis and function of miRNAs. (a) miRNAs are transcribed by RNA polymerase II as long primary trancripts, pri-miRNA. Upon transcription, the miRNAcontaining region of pre-miRNA forms a hairpin-loop, which is recognised by (b) the ‘‘microprocessor’’ complex composed of DGCR8 (blue oval) and Drosha (purple star). Drosha then cleaves the pre-miRNA hairpin-loop from the primary transcript, and (c) pre-miRNAs are exported from the nucleus by exportin 5. (d) Once in the cytoplasm, DICER 1 binds to pre-miRNA and the loop is removed, leaving a small double-stranded RNA. (e) The DICER 1-bound dsRNA then rapidly associates with the RNA-induced silencing complex (RISC), which is composed of the TAR RNA binding protein 2 (TRBP), an argonaute protein (AGO) and GW182, among others. Interaction with RISC leads to unwinding of the double stranded small RNA, and the miRNA strand (red strand) remains bound to AGO. The RISC bound miRNA is then able to carry out gene silencing. (f) RNA cleavage occurs in targets that display a fully complementary miRNA binding site and (g) translational repression occurs with partial complementarity between miRNA and its target.
the latter presumably through increased PDPK1 signaling (Figure 2c) [29]. These two studies highlight the importance of miRNAs in b-cell glucose responsiveness. Undoubtedly, future studies will focus on how other miRNAs regulate b-cell glucose sensing and how dysregulation of miRNA expression contributes to the loss of glucose competence and type 2 diabetes. Obesity leads to compensatory expansion of b-cell mass through increased b-cell replication. In type 2 diabetes, an increase in b-cell apoptosis concurrent with increased insulin demand leads to hyperglycemia. There has been substantial interest in understanding whether miRNAs regulate b-cell mass and whether they could be used as therapeutic agents for diabetes treatment. Lovis et al. [30] demonstrated that miRNA-34a is induced in a tumor protein p53-dependent manner in MIN6 B1 mouse b-cells by free fatty acids and in the db/db diabetic mouse model, and that miRNA-34a significantly increased apoptosis under normal conditions by reducing Bcl2 expression in MIN6 B1 cells. Thus, it is possible that therapeutic suppression of miRNA-34a could prevent b-cell loss during the development of diabetes [30]. In a recent study, Poy et al. reported the generation and phenotyping of the miRNA-375 knockout mouse (Figure 2d,f; Table 1) [32]. These animals display marked hyperglycemia that resulted from increased hepatic glucose output caused by increased a-cell mass and hyperglucago454
nemia, without an apparent increase in glucagon secretion per cell [32]. As in the earlier study [24], b-cell insulin secretory capacity was enhanced in these animals, but this increase was offset by a dramatic decrease in b-cell mass, attributed to increased expression of genes that negatively regulate b-cell replication (Figure 2d; Table 1) [32]. Because miRNA-375 expression was elevated in the ob/ob mouse [32], the miRNA-375 knockout was crossed into the ob/ob background to determine if miRNA-375 upregulation is important for the b-cell compensation observed in obesity. As early as 4 weeks of age, the double miRNA-375/leptin null animals (DKO) displayed significant hyperglycemia compared with either single knockout strain; by 10 weeks, the DKO showed significant reductions in b-cell replication and plasma insulin concomitant with increased hepatic glucose output [32]. It is unclear why a-cell replication increases whereas b-cell replication decreases in the miRNA-375 knockout animal (Figure 2f). It is hoped that future studies will address which genes are necessary for these effects in each cell type and clarify whether miRNA-375 loss results in a subtle but physiologically relevant increase in b-cell apoptosis. Liver miRNAs and lipid metabolism Once secreted from the islet, insulin and glucagon travel to the liver through the portal circulation where they control hepatic glucose and lipid metabolism. Liver insulin
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Figure 2. The role of miRNA-375 in pancreatic islet function. In the pancreatic b-cell (yellow), (a) miRNA-375 interacts with the myotrophin (Mtpn) mRNA leading to both down-regulation of mRNA expression and a decreased translation (red octagon). Knockdown of myotrophin results in suppression of insulin secretion possibly through the regulation of the actin cytoskeleton (F-Actin). (b) Insulin might regulate its own expression through binding to cell surface insulin receptors (INSR) and stimulating a phosphorylation-dependent signaling cascade that results in the phosphorylation of 3-phosphoinositide dependent protein kinase-1 (PDPK1). PDPK1 can then phosphorylate protein kinase B (AKT) and AKT, in turn, phosphorylates glycogen synthase kinase 3 beta (GSK3B), which phosphorylates the pancreatic and duodenal homeobox 1 (PDX1) transcription factor. Upon phosphorylation, pPDX1 translocates to the nucleus where it stimulates insulin transcription. (c) Pdpk1 is negatively regulated by miRNA-375, resulting in decreased insulin gene transcription. (d) Germline removal of miRNA-375 in mice results in the induction of several genes that are important for restraining cell replication; therefore, miRNA-375 is believed to normally stimulate expansion of b-cell mass through partial inhibition of these pathways. Glucose is important for b-cell replication, insulin gene transcription and insulin secretion. (e) Glucose represses expression of miRNA-375 through an undetermined mechanism thereby reversing all of these repressive effects (a–d). (f) The islet a-cell (blue) also expresses miRNA-375, and mice with null mutations in the miRNA-375 gene display markedly elevated plasma glucagon levels and increased a-cell proliferation. It is unclear which miRNA-375 target genes normally repress both a-cell replication and glucagon secretion. ?, unknown.
resistance plays an important role in the development of the metabolic syndrome. and dysregulation of miRNA expression potentially influences insulin resistance. Dicer1 deletion from the early postnatal liver resulted in profound hepatocyte apoptosis, steatosis and mild hypoglycemia [33]. The most highly expressed miRNA in the liver is miRNA122, which has been estimated to be expressed at up to 135,000 copies per human hepatocyte [34]. Several groups have assessed how miRNA-122 might affect hepatocyte biology by using antisense methods to suppress miRNA122 expression; all have shown that this miRNA is important for regulating liver lipid metabolism (Table 1) [35–38]. Mice treated with cholesterol-conjugated, 20 -O-methyl oligonucleotides antisense to miRNA-122 showed dramatic
reductions in peripheral expression of miRNA-122, with nearly complete loss in the liver [35,38]. This resulted in upregulation, at the message level, of a large number of genes that contained the miRNA-122 ‘‘seed’’ binding sequences in their 30 UTRs [38]. Knockdown of miRNA122 in mice decreased expression of several genes important for cholesterol biosynthesis, whereas adenoviral overexpression of miRNA-122 increased cholesterol biosynthesis. However, the effects of miRNA-122 on cholesterol biosynthesis are indirect, and it is unclear which direct targets of miRNA-122 mediate them [38]. A second complementary study found that miRNA-122 knockdown in mice decreased circulating cholesterol levels, liver cholesterol and fatty acid synthesis and increased liver fatty acid oxidation [37]. This 455
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Table 1. Target-miRNA pairs that have been experimentally validated as important for glucose and lipid metabolism Tissue/cell type Pancreatic Islet MIN6 mouse b-cells INS IE rat b-cells miR-375 knockout mouse islets
INS IE rat b-cells MIN6 Bl mouse b-cells MIN6 mouse b-cells MIN6 Bl mouse b-cells Liver primary mouse hepatocytes primary mouse hepatocytes primary mouse hepatocytes
miRNA
Biological Result of miRNA Expression
Putative Target Gene
Reference
miR-375
Suppression of insulin secretion Suppression of insulin transcription Cell growth, possibly through increased cell cycling Cell growth, possibly via reduced caspase-2 activity Cell growth, possibly via maintence of genomic stability Cell growth, via reduction in apoptosis Cell growth, via unknown mechanism Cell growth, possibly via increased Gi/Gq signalling Cell growth, possibly via reduction of p53 activity Cell growth, possibly via reduction in apoptosis Cell growth, possibly via increased cell cycling Cell growth, unknown mechanism Suppression of insulin secretion Dysregulation of insulin secretion Suppression of insulin transcription, possible increase in secretion Suppression of insulin secretion Sensitization to apoptosis
Mtpn Pdkl Cavl Id3 Smarca2 Aifml Rasdl Rgsl6 Eeflel Clqbp HuD Cadml Onecut2 Rab27A Foxa2 Vamp2 Bcl2
[24] [29] [32] [32] [32] [32] [32] [32] [32] [32] [32] [32] [26] [27] [28] [30] [30]
miR-122
Unknown, possibly reduced intracellular amino acid concentration Cholesterol synthesis, possibly reduced glycolysis Cholesterol synthesis, possibly reduced glycogenesis Synthesis, possibly increased cell cycling Cholesterol synthesis, possibly reduced RNAi Cholesterol synthesis, unknown role Cholesterol synthesis, unknown role Cholesterol synthesis, unknown role Cholesterol synthesis, unknown role Dysregulation of cholesterol synthesis and lipogenesis Dysregulation of cholesterol synthesis and lipogenesis Dysregulation cholesterol synthesis and lipogenesis Dysregulation cholesterol synthesis and lipogenesis
Slc7al Aldoa Gysl Ccngl P4hal Ndrg3 Tmed3 Hfe2 Slc35a4 SREBP2 HMGCR SREBPlc FAS
[34,37] [35,37,38] [37] [37] [37] [38] [38] [38] [38] [39] [39] [39] [39]
miR-278
Insulin sensitivity in Drosophila
expanded
[46]
miR-9 miR-124a miR-34a
primary mouse hepatocytes
HepG2 human hepatoma
Adipose
study also demonstrated that miRNA-122 inhibition reduced both hepatic cholesterol accumulation and liver steatosis during the development of diet-induced obesity in mice. Finally, these authors demonstrated an increase in phosphorylated AMP-activated protein kinase (AMPK) in livers of these mice, but failed to clarify if miRNA-122 was directly regulating AMPK signaling [37]. A third study in primates found that inhibition of miRNA-122 with antisense molecules reduced plasma cholesterol levels without any apparent liver toxicity [36], suggesting miRNA inhibition as a feasible therapeutic approach in humans. miRNA-122 has been demonstrated to be important for liver fat metabolism, but there is also some evidence that dysregulation of miRNA expression might underlie some disease pathologies [39]. Nonalcoholic fatty liver disease (NAFLD) has been demonstrated to be present in a large percentage of individuals with metabolic syndrome, and might be partly responsible for the development of liver insulin resistance [40]. In one study, 46 miRNAs were differentially expressed in humans with NAFLD [39]. miRNA-122 was in the group of genes that was downregulated in NAFLD, and this was correlated with increased expression of lipogenic genes in the human livers. Knockdown of miRNA-122 in HepG2 cells recapitulated the lipogenic gene expression profile observed in individuals with NAFLD [39]. In this case, it seems likely that miRNA-122 downregulation is a compensatory mechanism that counters increasing liver lipid levels, rather 456
than a causative agent in the development of NAFLD. Future studies should clarify this apparent inconsistency. In any case, the future use of miRNA-122 mimetics or antagonists could rebalance the defects observed in the liver during metabolic syndrome [41]. Adipose tissue miRNAs and energy metabolism Fatty acids travel in lipoprotein particles from the liver to the adipose tissue where they are stored in lipid droplets. Knockdown of either DICER1 or RNASEN prevents in vitro differentiation of human multipotent stromal cells to adipocytes, implying that miRNAs might be important for adipogenesis [42]. The first indication that miRNAs might be important in adipose cell biology came from the observation that miRNA-143 is one of several miRNAs that are upregulated during differentiation of human pre-adipocytes [43]. Inhibition of miRNA-143 action by antisense oligonucleotides led to a substantial inhibition of differentiation with decreases in triglyceride accumulation, and reductions in expression of the adipocyte-specific genes fatty acid binding protein 4 (FABP4), glucose transporter 4 (SLC2A4), peroxisome proliferator activated receptor gamma (PPARG) and hormone sensitive lipase (LIPE) [43]. Mitogen-activated protein kinase 7 (MAPK7) was shown to be upregulated by knockdown of miRNA-143 in these experiments but its role in adipogenesis is presently unclear [43]. Xie et al. found that both miRNA-143 and -103 were upregulated during adipogenesis in mouse 3T3-L1
Review preadipocytes [44]. Furthermore, overexpressing these two miRNAs during adipogenesis resulted in significantly increased expression of several adipocyte-specific genes. These two miRNAs are markedly downregulated, possibly through an inflammatory pathway in the development of obesity in both ob/ob mice and mice fed a high-fat diet [44]. The first report implicating miRNA involvement in adipose-regulated energy metabolism was carried out using Drosophila. miRNA-14 null adult flies have substantially increased lipid droplet accumulation in adipose tissue. In addition, whole body increases in both di- and triacylglycerol levels were observed, and these were correlated with increased apoptosis in several tissues in the mutant flies [45]. A second study in flies demonstrated that miRNA-278 mutants had reduced whole body fat levels [46], yet these mutant flies also displayed elevated insulin and sugar levels, primarily in the form of trehalose, which is generated from mobilisation of glycogen reserves in the Drosophila adipose tissue analog. The observation of elevated sugars and loss of body fat in the face of elevated insulin levels suggests that the fat body in the miRNA-278 mutant is resistant to insulin [46]. This hypothesis was confirmed by the observations that expression levels of the insulin target genes 4E-BP and InR were inappropriately high in the mutant fly fat bodies. Overexpression of the putative miRNA-278 target gene expanded (ex), which can act as a tumor suppressor, was able to phenocopy the miRNA-278 knockout, suggesting that ex is a likely biological target of miRNA-278 in Drosophila (Table 1) [46]. Although neither miRNA-278 nor miRNA-14 is expressed in higher organisms, similar phenotypes are observed upon loss of insulin signaling in adipose tissue in mammals. For example, mice that have the insulin receptor gene selectively deleted from adipose tissue are lean and have a reduction in fat pad mass, whereas mice that have the insulin receptor gene removed from muscle show increased adipose tissue stores [47,48]. One study has directly addressed the role of miRNA-29 in the development of insulin resistance in mammals. This study demonstrated that miRNA-29a, b and c were all upregulated in adipose tissue of diabetic rats, and that this upregulation could be induced by exposing 3T3-L1 mouse adipocytes to insulin and glucose [49]. Overexpressing miRNA-29 in the same adipocytes reduced insulin-stimulated glucose uptake, a reduction that was attributed to disrupted insulin signaling through a reduction in insulin-stimulated AKT1 phosphorylation. However, it remains unclear which miRNA-29 target genes mediate these effect in adipocytes [49]. Adipocytes can be functionally characterized into two groups: energy-storing white adipocytes and energyexpending brown adipocytes. Recently, it was demonstrated that brown adipocytes and muscle share a common precursor, and that the zinc finger transcription factor PRDM16 regulates the differentiation of brown fat at the expense of muscle [50]. In line with their common embryological origins, brown adipose tissue and muscle share similar patterns of miRNA expression, with low levels of miRNA-143 and higher levels of miRNA-1, -133a and -206 [51]. The physiological significance of this expression profile, however, has not yet been addressed.
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Future studies might be targeted at understanding how additional miRNAs regulate adipocyte insulin sensitivity, adipokine and adipose-derived hormone secretion, and lipolysis both normally and in disease. Muscle and brain miRNAs and metabolic control Muscle is a major site of glucose disposal and metabolism. Although there is substantial evidence demonstrating a role for miRNAs in muscle growth and development [52], little is known about miRNA regulation of muscle glucose metabolism. Dicer1 removal from cardiac progenitors, from adult hearts and from developing muscle all lead to tissue loss and death [53–55]. The miRNA-1/206 and the miRNA133a/133b families appear to be most important for muscle development and growth. These miRNAs are all regulated by one or more of the myogenic transcription factors, and are important for specification and function of both cardiac and skeletal muscle [52]. It has been demonstrated that some miRNAs are differentially expressed in the skeletal muscle of diabetic GK rats; however, the physiological relevance of this observation is unknown [49]. It is hoped that future studies will focus on understanding whether miRNAs can regulate insulin signaling in muscle and whether ‘‘myomiR’’ levels are dysregulated during metabolic disturbances and pathologies. Circulating nutrients and hormones not only affect peripheral tissues but also the feeding and satiety centers in the hypothalamus; in turn, afferent signals from the brain regulate pancreatic islet, liver and adipose physiology [56]. Thus, the brain should be considered part of the system responsible for the regulation of carbohydrate and fat metabolism. A plethora of miRNAs is expressed in the nervous system, and removal of Dicer1 in specific brain regions can lead to both behavioral defects and neurodegeneration [57–59]. The role of miRNAs in the feeding and satiety centers has not been addressed; however, as in all the other metabolically active tissues, they undoubtedly play a role in these neurons as well. Concluding remarks It is now clear that gene regulatory mechanisms governed by miRNAs are important for the development and maintenance of metabolically active tissues. Furthermore, alteration in miRNA expression in several of these tissues can result in impaired glucose and lipid homeostasis through a wide range of mechanisms ranging from cell cycle control to exocytotic regulation. Unfortunately, the regulatory power that miRNAs exert through their large repertoire of target genes is the Achilles heel of miRNA research, as it is difficult to dissect out the specific factors that are important for any one phenotype. In the future, it will be important to develop experimental methods that enable a more integrative approach to miRNA biology including hierarchical assignment of target relevance, regulation of tissue-specific miRNA expression and processing during disease, development of methods for the facile generation of multiallelic knockouts to study miRNA families, and novel delivery methods for tissuespecific therapeutics. These tiny regulators are clearly important for normal biology and in disease etiology, and their regulation of regulators of glucose and lipid 457
Review metabolism or ‘‘meta-regulation’’ clearly warrants further research. Acknowledgements I am indebted to Michael German, Matthias Hebrok and Michael McManus for their help, support and advice. Postdoctoral fellowship awards 3-2004-276 and 10-2007-86 from Juvenile Diabetes Research Foundation allowed me to carry out studies in this field and for this I am grateful. This is a burgeoning field and as such there has been a huge increase in the number of publications over the past 5 years. This large number of exciting publications has made this article possible; however, the length of this review precludes citation of original manuscripts and I apologise for this.
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