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Roles of Micro-RNAs in Metabolism
Roles of Micro-RNAs in Metabolism Z Wei and G W Wong, Johns Hopkins Univesity School of Medicine, Baltimore, MD, USA ã 2013 Elsevier Inc. All rights reserved.
Glossary Adipocyte differentiation The process through which adipocyte precursor cells acquire characteristics of mature adipocytes. Antagomir Antisense oligonucleotide sequence conjugated to cholesterol. Antisense oligonucleotides Single strands of DNA or RNA that are complementary to a chosen messenger RNA sequence.
Introduction Transcriptional regulation of gene expression represents one of several important and fundamental mechanisms governing energy metabolism. There are many different classes of RNAs, one of which is called micro-RNAs (miRNA). miRNAs are small, noncoding RNAs 19–22 nucleotides in length. They regulate gene expression post-transcriptionally by base-pairing with their cognate target sequences located at the 30 -untranslated regions (30 -UTRs) of protein-coding mRNAs, leading to cleavage or translational inhibition of the target mRNAs. This miRNA-based mechanism of regulating gene expression is evolutionarily conserved from plants to humans. Abundant and ubiquitously expressed, this class of small RNAs has emerged as an important tool kit employed by cells to fine-tune the expression levels of protein-coding genes. As many as 721, 579, and 325 miRNAs have been discovered in the human, mouse, and rat genomes, respectively; annotations of these miRNAs have been deposited in a public miRNA repository database, miRBase. Estimates suggest that 1–3% of the human and mouse genomes code for miRNA genes, and as much as 30% of the protein-coding genes are regulated by miRNAs in higher eukaryotic species. Therefore, it is not surprising that miRNAs have been shown to play wide-ranging roles in many biological processes, including development, cell differentiation, apoptosis, and immune response. Rapid progress has also been made recently to understand the role of miRNAs in metabolism. Recent studies suggest that miRNAs participate in metabolic processes, including insulin secretion, pancreatic islet cell development, adipocyte differentiation, and insulin signaling, in a variety of tissues. The discovery of miRNAs and their roles in regulating metabolism has added a new dimension to our understanding of the complex metabolic circuitry that governs energy homeostasis.
miRNA Biogenesis Approximately 720 miRNA coding regions are scattered throughout the human genome. Some miRNA genes exist as independent transcriptional units and are often located in
Knockdown The reduction of gene expression achieved by certain techniques, such as RNA interference. Knockout The deletion of gene(s) from the genome of an organism. b-Cell A type of cell found in the islets of Langerhans in pancreas that produces insulin.
regions of the genome devoid of protein-coding genes. Other miRNA genes are located in the intronic regions of proteincoding genes. The biogenesis of miRNA starts with transcription initiated by RNA polymerase II or III, generating a long primary miRNA (pri-miRNA) transcript containing a 50 -cap structure and a 30 -poly(A) tail (Figure 1). The complementary region of the pri-miRNA forms a hairpin loop. The loop is recognized by the miRNA processing machinery, composed of Drosha and DGCR8 (DiGeorge syndrome critical region protein 8), which cleaves the structure at the bottom of the stem loop to generate an 70-nucleotide precursor miRNA (pre-miRNA) transcript. The pre-miRNAs are then exported from the nucleus by the nuclear export machinery (exportin 5 and Ran-GTP). Once in the cytoplasm, the pre-miRNAs are further processed by Dicer, an RNase III-type enzyme, to generate 22-nucleotide miRNA duplexes. These RNA duplexes are subsequently incorporated into the RNA-induced silencing complex (RISC). One strand of the RNA duplex is discarded while the mature miRNA strand remains in the RISC. The miRNAs then mediate their effects on gene expression by base-pairing with complementary sequences in the 30 -UTR of the target mRNAs, resulting in silencing of the target mRNAs via Argonaut-dependent mRNA cleavage and/or translational repression. Translationally repressed mRNAs are either degraded by the mRNA decay pathway or stored away in a specialized intracellular granule compartment termed the ‘P-body’.
miRNA in Pancreatic b-Cell Biology The evidence for miRNA functions in pancreatic b-cells came from the cloning of small RNAs found in the mouse insulinoma cell line, MIN6. The most abundant miRNA in MIN6 cells is miR-375, which has been shown to negatively regulate glucosestimulated insulin secretion. Overexpression of miR-375 leads to decreased glucose-stimulated insulin release, while loss of function of miR-375 increases insulin release from pancreatic b-cells. miR-375 regulates insulin secretion partly by repressing the expression of myotrophin (Mtpn), a protein important for intracellular vesicle transport in neurons. Indeed, overexpression of miR-375 in cells decreases the expression of
191
192
Metabolism Vitamins and Hormones | Roles of Micro-RNAs in Metabolism
RNA Pol II/III miRNA gene Nucleus
Cytoplasm
DGCR8
Drosha 5⬘Cap
Pre-miRNA
miRNA duplex
Poly(A)3⬘ Pri-miRNA Dicer
Ago
RISC
Exportin 5 Pre-miRNA
Discarded Target mRNA
Ribosome
RISC
Translation repression
Mature miRNA
RISC
miRNA cleavage
Figure 1 The biogenesis of miRNA.
Mtpn. miR-375 binds directly to the 30 -UTR region of the Mtpn gene, indicating that Mtpn is a direct target of miR-375. Importantly, knockdown of Mtpn mRNA levels by small interfering RNAs (siRNA) also inhibits glucose-stimulated insulin release, consistent with the inhibitory effect of miR-375 on insulin secretion. miR-9 also negatively regulates insulin secretion, by repressing the expression of a transcriptional repressor, Onecut2 (OC2). One of the genes repressed by OC2 is the guanosine triphosphatase (GTPase) effector, granuphilin/Slp4. Granuphilin is associated with secretory granules and, upon glucose stimulation, negatively regulates the exocytosis of insulin-containing granules from the b-cells. Therefore, when the expression of OC2 is inhibited by miR-9, the expression levels of granuphilin/Slp4 increase. Increased granuphilin/Slp4 results in decreased glucose-stimulated insulin secretion from the b-cells. Because OC2 is a direct target of miR-9, silencing OC2 expression by RNAi mimics the effects of miR-9 on the expression of granuphilin/Slp4 and insulin-containing granules exocytosis. miRNAs also play a role in the development and proliferation of insulin-producing pancreatic b-cells. For example, miR124a is abundantly expressed in b-cells and inhibits b-cell development. It acts by targeting Foxa2, a critical transcription factor that regulates b-cell development. Overexpression of miR-124a inhibits the expression of Foxa2 and other essential genes (e.g., Pdx-1, Kir6.2, and Sur-1) involved in b-cell development and function. Knockdown of miR-124a levels in cells abolishes the inhibitory effects on Foxa2 and other targets. In contrast to miR-124, miR-375 positively promotes the development and/or proliferation of b-cells. Mice in which the miR-375 gene has been ablated show significant increases in the numbers of glucagon-producing a-cells, and a decrease in
the numbers of insulin-producing b-cells in their pancreatic islets. These studies support a role for miR-375 in regulating different endocrine cell types in the pancreas.
miRNA in Lipid Metabolism A study demonstrating how a specific miRNA, miR-14, regulates lipid metabolism in the fruit fly, Drosophila melanogaster, first implicated miRNAs in this process. miR-14 negatively regulates intracellular triglyceride levels in the fat body (analogous to mammalian adipose tissue) of Drosophila. Ablating the miR-14 gene in Drosophila increases triacylglycerol and diacylglyerol levels; however, increasing miR-14 expression lowers these levels. Similarly, miR-278 has been shown to regulate fat body metabolism in Drosophila. Loss of function of miR-278 results in decreased fat body size and a corresponding reduction in whole-body triglyceride-to-protein ratio in flies. Interestingly, miR-278-deficient flies also suffer from hyperglycemia despite increased levels of insulin-like peptide, suggesting the presence of insulin resistance in these flies. Although mammals do not have the equivalent of Drosophila miR-14 or miR-278, miRNAs are involved in regulating mammalian lipid metabolism. For example, miR-143 promotes adipogenesis. The expression of miR-143 increases during the course of adipogenesis in tissue culture, as well as in mice. Knockdown of miR-143 levels in human preadipocytes by antisense oligonucleotides causes a significant decrease in adipocyte differentiation. Conversely, overexpression of miR-143 in murine 3T3-L1 preadipocytes accelerates differentiation into mature adipocytes. Similarly, miR-103 promotes adipocyte differentiation. Overexpressing miR-103 in 3T3-L1
Metabolism Vitamins and Hormones | Roles of Micro-RNAs in Metabolism
preadipocytes leads to increased expression of many adipocytespecific markers, such as peroxisome proliferator-activated receptor-g (PPAR-g) and fatty acid binding protein 4 (FABP4), concurrent with an early increase in triglyceride accumulation during the course of adipocyte differentiation in culture. Two additional miRNA families, miR-200 and miR-17-92, also positively regulate the process of adipocyte development. Overexpression of the miR-200 or miR-17-92 clusters accelerates the conversion of mouse ST2 marrow stromal cells or 3T3-L1 fibroblasts into mature adipocytes, respectively. In contrast, one particular miRNA, miR-27, opposes adipocyte differentiation by targeting and inhibiting the expression of two key transcription factors that drive adipogenesis, PPAR-g and CCAAT/ enhancer binding protein-alpha (C/EBP-a). These recent studies highlight the intersection of transcription- and miRNA-based mechanisms in controlling adipocyte differentiation. Unlike in adipocytes, liver miRNA expression is dominated by a single miRNA, miR-122, which exists in 50 000 copies per cell. Abolishment of miR-122 expression in mice using antisense oligonucleotides conjugated to cholesterol (called antagomirs) results in the lowering of plasma cholesterol levels. This effect is mediated, in part, by the miR-122-dependent repression of cholesterol biosynthetic genes in liver, such as the HMG-CoA reductase. A second study, using a similar knockdown approach against miR-122, demonstrated a reduction in fatty acid synthesis and an increase in fatty acid oxidation in primary hepatocytes. These metabolic alterations are accompanied by an increase in adenosine monophosphate (AMP)-activated kinase (AMPK) levels, consistent with the known function of AMPK in promoting fatty acid oxidation while inhibiting fatty acid synthesis in liver.
miRNA in Glucose Metabolism and Insulin Signaling miRNAs can not only regulate glucose metabolism by affecting the magnitude of insulin secretion from pancreatic b-cells, but also modulate insulin signaling and action in insulinresponsive tissues. Studies comparing miRNA expression profiles in healthy versus type 2 diabetic rats identified two miR-29 family members, miR-29a and miR-29b, that are upregulated in muscle, adipose, and liver of diabetic animals. Overexpression of miR-29 in 3T3-L1 adipocytes suppresses insulinstimulated glucose uptake, an effect likely due to inhibition of protein kinase B/Akt activation. Consistent with its upregulation in diabetic animals, miR-29 expression is also increased in adipocytes cultured in high glucose and high insulin-containing media. This observation suggests a possible involvement of miR-29 in the development of insulin resistance; however, the mechanism behind the role of miR-29 in insulin resistance is not known. Although insulin-induced gene 1 (Insig1) and caveolin 2 (Cav2) were identified as miR-29 targets, their roles in insulin signaling and glucose metabolism remain to be clarified. Other miRNAs are also implicated in controlling the insulin receptor signaling pathway. However, the direct effects of these miRNAs on glucose metabolism remain to be established. For example, miR-145 can directly target and suppress the expression of insulin receptor substrate 1 (IRS1), a key component of the insulin receptor signaling pathway. Overexpression of miR-145 in colon cancer cells leads to growth arrest, a
193
phenotype reminiscent of IRS1 knockdown by siRNA in these cells. Another miRNA cluster, miR-183-96-182, targets multiple components of the insulin receptor signaling pathway, including IRS1, Rasa1, and Grb2. These observations suggest that for a given signaling pathway, multiple miRNAs can target different components of the signaling cascade, effectively shutting it off.
miRNA in the Treatment of Metabolic Diseases The implication of specific miRNAs in metabolic diseases (e.g., miR-29 in diabetes) has raised the hope that targeting these miRNAs may be a viable therapeutic approach to treat metabolic disorders. Various methods and tools are now available to modulate the expression of specific miRNAs in vivo. A gain of function can be achieved in vivo using viral-based methods to overexpress a synthetic form of any given miRNA in a desired tissue. A loss of function can be achieved in vivo by the delivery of antisense oligonucleotides specific to a target miRNA. In general, these antisense oligonucleotides are chemically modified to increase their stability and binding specificity. Several proof-of-principle studies have successfully demonstrated the feasibility and effectiveness of using adenovirus and lentivirus to deliver miRNA genes in mice. In addition to viral-based strategies, synthetic RNAs (e.g., antisense oligonucleotides) can be successfully delivered to tissues in vivo by conjugating synthetic RNAs to cholesterol and encapsulating them within the high- or low-density lipoproteins. However, this approach is limited because the encapsulated synthetic RNAs can be best delivered to only a few tissues such as the liver, intestine, and kidney. Perhaps a bigger problem with targeting miRNA to treat diseases lies in the specificity of the miRNA therapy. First, one miRNA can target several distinct genes and one gene can be targeted by multiple distinct miRNAs. It is not desirable to suppress those miRNAs that are not directly involved in disease pathogenesis. Thus, the potential off-target effects pose a significant challenge to miRNA-based therapy. Second, most of the miRNAs exhibit spatial, temporal, and tissue-specific expression patterns, characteristics necessary to their proper function. Therefore, a generic overexpression of miRNAs may not achieve the desired therapeutic effects. Whether miRNA-based therapeutic agents can be successfully deployed to treat metabolic diseases in clinical settings remains an open question.
Conclusion miRNAs comprise a diverse family of small noncoding RNAs that play important roles in fine-tuning gene expression and have been implicated in many fundamental physiologic processes. The roles of miRNA in metabolism (Table 1) are still being uncovered, and many miRNAs potentially important to this process are yet to be characterized. The immediate goals in the miRNA community are to validate the authenticity of the predicted miRNA genes in vivo and to clone new miRNA genes, establish their function, and identify their mRNA targets. While miRNA-based therapy is an attractive alternative to treat certain metabolic diseases, the questionable specificity of
194
Table 1
Metabolism Vitamins and Hormones | Roles of Micro-RNAs in Metabolism
The role of miRNAs in metabolism
miRNA
Metabolic regulation
Target tissues/cell lines
Target genes
miR375 miR9 miR-124a miR-14 miR-278 miR-143 miR-103 miR-200 miR-17-92 miR-27 miR-122 miR-29 miR-145 miR-183-96-182
Insulin secretion; a- and b-cell proliferation; b-cell development Insulin secretion Development and proliferation of b-cell Lipid metabolism in fat body Lipid metabolism in fat body; insulin resistance Adipogenesis Adipogenesis Adipogenesis Adipogenesis Adipogenesis Cholesterol and fatty acid metabolism Diabetes, insulin resistance Insulin signaling; growth Insulin signaling
Pancreas Pancreas Pancreas Fat body (Drosophila) Fat body (Drosophila) Human preadipocytes; 3T3-L1 cells 3T3-L1 Mouse ST2 cells 3T3-L1cells 3T3-L1 cells Liver Muscle, adipose, and liver Colon cancer cells
Mtpn, Usp1 OC2 Foxa2, Rab27A Unknown Expanded ERK5/MAPK7 Unknown Unknown Rb2/p130 PPAR-g and C/EBP-a HMG-CoA reductase Insig1 and Cav2 IRS1 IRS1, Rasa1, and Grb2
miRNA-based therapy and the challenge of delivering miRNA or anti-miRNA molecules to the desired target tissues in vivo will be the key issues that need to be addressed in the near future.
See also: Molecular Biology: MicroRNAs in Eukaryotes.
Further Reading Baroukh N, Ravier MA, Loder MK, et al. (2007) MicroRNA-124a regulates Foxa2 expression and intracellular signaling in pancreatic beta-cell lines. Journal of Biological Chemistry 282(27): 19575–19588. Bartel DP (2009) MicroRNAs: Target recognition and regulatory functions. Cell 136(2): 215–233. Esau C, Davis S, Murray SF, et al. (2006) miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metabolism 3(2): 87–98. Esau C, Kang X, Peralta E, et al. (2004) MicroRNA-143 regulates adipocyte differentiation. Journal of Biological Chemistry 279(50): 52361–52365. Gauthier BR and Wollheim CB (2006) MicroRNAs: ‘Ribo-regulators’ of glucose homeostasis. Nature Medicine 12(1): 36–38. He A, Zhu L, Gupta N, Chang Y, and Fang F (2007) Overexpression of mir-29, highly up-regulated in diabetic rats, leads to insulin resistance in 3T3-L1 adipocytes. Molecular Endocrinology 21(11): 2785–2794. Kru¨tzfeldt J, Rajewsky N, Braich R, et al. (2005) Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438(7068): 685–689.
Lin Q, Gao Z, Alarcon RM, Ye J, and Yun Z (2009) A role of miR-27 in the regulation of adipogenesis. FEBS Journal 276(8): 2348–2358. Lynn FC (2009) Meta-regulation: MicroRNA regulation of glucose and lipid metabolism. Trends in Endocrinology and Metabolism 20(9): 452–459. Pandey AK, Agarwal P, Kaur K, and Datta M (2009) MicroRNAs in diabetes: Tiny players in big disease. Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology 23(4–6): 221–232. Poy MN, Eliasson L, Krutzfeldt J, et al. (2004) A pancreatic islet-specific microRNA regulates insulin secretion. Nature 432(7014): 226–230. Poy MN, Hausser J, Trajkovski M, et al. (2009) miR-375 maintains normal pancreatic alpha- and beta-cell mass. Proceedings of the National Academy of Sciences of the United States of America 106(14): 5813–5818. Poy MN, Spranger M, and Stoffel M (2009) MicroRNAs and the regulation of glucose and lipid metabolism. Diabetes, Obesity and Metabolism 9(supplement 2): 67–73. Tang X, Tang G, and Ozcan S (2008) Role of microRNAs in diabetes. Biochimica et Biophysica Acta 1779(11): 697–701. Teleman AA, Maitra S, and Cohen SM (2006) Drosophila lacking microRNA miR-278 are defective in energy homeostasis. Genes and Development 20(4): 417–422. Wang Q, Li YC, Wang J, et al. (2008) miR-17-92 cluster accelerates adipocyte differentiation by negatively regulating tumor-suppressor Rb2/p130. Proceedings of the National Academy of Sciences of the United States of America 105(8): 2889–2894. Xie H, Lim B, and Lodish HF (2009) MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity. Diabetes 58(5): 1050–1057. Xie H, Sun L, and Lodish HF (2009) Targeting microRNAs in obesity. Expert Opinion on Therapeutic Targets 13(10): 1227–1238.
Glossary Adipocyte differentiation The process through which adipocyte precursor cells acquire characteristics of mature adipocytes. Antagomir Antisense oligonucleotide sequence conjugated to cholesterol. Antisense oligonucleotides Single strands of DNA or RNA that are complementary to a chosen messenger RNA sequence.
Introduction Transcriptional regulation of gene expression represents one of several important and fundamental mechanisms governing energy metabolism. There are many different classes of RNAs, one of which is called micro-RNAs (miRNA). miRNAs are small, noncoding RNAs 19–22 nucleotides in length. They regulate gene expression post-transcriptionally by base-pairing with their cognate target sequences located at the 30 -untranslated regions (30 -UTRs) of protein-coding mRNAs, leading to cleavage or translational inhibition of the target mRNAs. This miRNA-based mechanism of regulating gene expression is evolutionarily conserved from plants to humans. Abundant and ubiquitously expressed, this class of small RNAs has emerged as an important tool kit employed by cells to fine-tune the expression levels of protein-coding genes. As many as 721, 579, and 325 miRNAs have been discovered in the human, mouse, and rat genomes, respectively; annotations of these miRNAs have been deposited in a public miRNA repository database, miRBase. Estimates suggest that 1–3% of the human and mouse genomes code for miRNA genes, and as much as 30% of the protein-coding genes are regulated by miRNAs in higher eukaryotic species. Therefore, it is not surprising that miRNAs have been shown to play wide-ranging roles in many biological processes, including development, cell differentiation, apoptosis, and immune response. Rapid progress has also been made recently to understand the role of miRNAs in metabolism. Recent studies suggest that miRNAs participate in metabolic processes, including insulin secretion, pancreatic islet cell development, adipocyte differentiation, and insulin signaling, in a variety of tissues. The discovery of miRNAs and their roles in regulating metabolism has added a new dimension to our understanding of the complex metabolic circuitry that governs energy homeostasis.
miRNA Biogenesis Approximately 720 miRNA coding regions are scattered throughout the human genome. Some miRNA genes exist as independent transcriptional units and are often located in
Knockdown The reduction of gene expression achieved by certain techniques, such as RNA interference. Knockout The deletion of gene(s) from the genome of an organism. b-Cell A type of cell found in the islets of Langerhans in pancreas that produces insulin.
regions of the genome devoid of protein-coding genes. Other miRNA genes are located in the intronic regions of proteincoding genes. The biogenesis of miRNA starts with transcription initiated by RNA polymerase II or III, generating a long primary miRNA (pri-miRNA) transcript containing a 50 -cap structure and a 30 -poly(A) tail (Figure 1). The complementary region of the pri-miRNA forms a hairpin loop. The loop is recognized by the miRNA processing machinery, composed of Drosha and DGCR8 (DiGeorge syndrome critical region protein 8), which cleaves the structure at the bottom of the stem loop to generate an 70-nucleotide precursor miRNA (pre-miRNA) transcript. The pre-miRNAs are then exported from the nucleus by the nuclear export machinery (exportin 5 and Ran-GTP). Once in the cytoplasm, the pre-miRNAs are further processed by Dicer, an RNase III-type enzyme, to generate 22-nucleotide miRNA duplexes. These RNA duplexes are subsequently incorporated into the RNA-induced silencing complex (RISC). One strand of the RNA duplex is discarded while the mature miRNA strand remains in the RISC. The miRNAs then mediate their effects on gene expression by base-pairing with complementary sequences in the 30 -UTR of the target mRNAs, resulting in silencing of the target mRNAs via Argonaut-dependent mRNA cleavage and/or translational repression. Translationally repressed mRNAs are either degraded by the mRNA decay pathway or stored away in a specialized intracellular granule compartment termed the ‘P-body’.
miRNA in Pancreatic b-Cell Biology The evidence for miRNA functions in pancreatic b-cells came from the cloning of small RNAs found in the mouse insulinoma cell line, MIN6. The most abundant miRNA in MIN6 cells is miR-375, which has been shown to negatively regulate glucosestimulated insulin secretion. Overexpression of miR-375 leads to decreased glucose-stimulated insulin release, while loss of function of miR-375 increases insulin release from pancreatic b-cells. miR-375 regulates insulin secretion partly by repressing the expression of myotrophin (Mtpn), a protein important for intracellular vesicle transport in neurons. Indeed, overexpression of miR-375 in cells decreases the expression of
191
192
Metabolism Vitamins and Hormones | Roles of Micro-RNAs in Metabolism
RNA Pol II/III miRNA gene Nucleus
Cytoplasm
DGCR8
Drosha 5⬘Cap
Pre-miRNA
miRNA duplex
Poly(A)3⬘ Pri-miRNA Dicer
Ago
RISC
Exportin 5 Pre-miRNA
Discarded Target mRNA
Ribosome
RISC
Translation repression
Mature miRNA
RISC
miRNA cleavage
Figure 1 The biogenesis of miRNA.
Mtpn. miR-375 binds directly to the 30 -UTR region of the Mtpn gene, indicating that Mtpn is a direct target of miR-375. Importantly, knockdown of Mtpn mRNA levels by small interfering RNAs (siRNA) also inhibits glucose-stimulated insulin release, consistent with the inhibitory effect of miR-375 on insulin secretion. miR-9 also negatively regulates insulin secretion, by repressing the expression of a transcriptional repressor, Onecut2 (OC2). One of the genes repressed by OC2 is the guanosine triphosphatase (GTPase) effector, granuphilin/Slp4. Granuphilin is associated with secretory granules and, upon glucose stimulation, negatively regulates the exocytosis of insulin-containing granules from the b-cells. Therefore, when the expression of OC2 is inhibited by miR-9, the expression levels of granuphilin/Slp4 increase. Increased granuphilin/Slp4 results in decreased glucose-stimulated insulin secretion from the b-cells. Because OC2 is a direct target of miR-9, silencing OC2 expression by RNAi mimics the effects of miR-9 on the expression of granuphilin/Slp4 and insulin-containing granules exocytosis. miRNAs also play a role in the development and proliferation of insulin-producing pancreatic b-cells. For example, miR124a is abundantly expressed in b-cells and inhibits b-cell development. It acts by targeting Foxa2, a critical transcription factor that regulates b-cell development. Overexpression of miR-124a inhibits the expression of Foxa2 and other essential genes (e.g., Pdx-1, Kir6.2, and Sur-1) involved in b-cell development and function. Knockdown of miR-124a levels in cells abolishes the inhibitory effects on Foxa2 and other targets. In contrast to miR-124, miR-375 positively promotes the development and/or proliferation of b-cells. Mice in which the miR-375 gene has been ablated show significant increases in the numbers of glucagon-producing a-cells, and a decrease in
the numbers of insulin-producing b-cells in their pancreatic islets. These studies support a role for miR-375 in regulating different endocrine cell types in the pancreas.
miRNA in Lipid Metabolism A study demonstrating how a specific miRNA, miR-14, regulates lipid metabolism in the fruit fly, Drosophila melanogaster, first implicated miRNAs in this process. miR-14 negatively regulates intracellular triglyceride levels in the fat body (analogous to mammalian adipose tissue) of Drosophila. Ablating the miR-14 gene in Drosophila increases triacylglycerol and diacylglyerol levels; however, increasing miR-14 expression lowers these levels. Similarly, miR-278 has been shown to regulate fat body metabolism in Drosophila. Loss of function of miR-278 results in decreased fat body size and a corresponding reduction in whole-body triglyceride-to-protein ratio in flies. Interestingly, miR-278-deficient flies also suffer from hyperglycemia despite increased levels of insulin-like peptide, suggesting the presence of insulin resistance in these flies. Although mammals do not have the equivalent of Drosophila miR-14 or miR-278, miRNAs are involved in regulating mammalian lipid metabolism. For example, miR-143 promotes adipogenesis. The expression of miR-143 increases during the course of adipogenesis in tissue culture, as well as in mice. Knockdown of miR-143 levels in human preadipocytes by antisense oligonucleotides causes a significant decrease in adipocyte differentiation. Conversely, overexpression of miR-143 in murine 3T3-L1 preadipocytes accelerates differentiation into mature adipocytes. Similarly, miR-103 promotes adipocyte differentiation. Overexpressing miR-103 in 3T3-L1
Metabolism Vitamins and Hormones | Roles of Micro-RNAs in Metabolism
preadipocytes leads to increased expression of many adipocytespecific markers, such as peroxisome proliferator-activated receptor-g (PPAR-g) and fatty acid binding protein 4 (FABP4), concurrent with an early increase in triglyceride accumulation during the course of adipocyte differentiation in culture. Two additional miRNA families, miR-200 and miR-17-92, also positively regulate the process of adipocyte development. Overexpression of the miR-200 or miR-17-92 clusters accelerates the conversion of mouse ST2 marrow stromal cells or 3T3-L1 fibroblasts into mature adipocytes, respectively. In contrast, one particular miRNA, miR-27, opposes adipocyte differentiation by targeting and inhibiting the expression of two key transcription factors that drive adipogenesis, PPAR-g and CCAAT/ enhancer binding protein-alpha (C/EBP-a). These recent studies highlight the intersection of transcription- and miRNA-based mechanisms in controlling adipocyte differentiation. Unlike in adipocytes, liver miRNA expression is dominated by a single miRNA, miR-122, which exists in 50 000 copies per cell. Abolishment of miR-122 expression in mice using antisense oligonucleotides conjugated to cholesterol (called antagomirs) results in the lowering of plasma cholesterol levels. This effect is mediated, in part, by the miR-122-dependent repression of cholesterol biosynthetic genes in liver, such as the HMG-CoA reductase. A second study, using a similar knockdown approach against miR-122, demonstrated a reduction in fatty acid synthesis and an increase in fatty acid oxidation in primary hepatocytes. These metabolic alterations are accompanied by an increase in adenosine monophosphate (AMP)-activated kinase (AMPK) levels, consistent with the known function of AMPK in promoting fatty acid oxidation while inhibiting fatty acid synthesis in liver.
miRNA in Glucose Metabolism and Insulin Signaling miRNAs can not only regulate glucose metabolism by affecting the magnitude of insulin secretion from pancreatic b-cells, but also modulate insulin signaling and action in insulinresponsive tissues. Studies comparing miRNA expression profiles in healthy versus type 2 diabetic rats identified two miR-29 family members, miR-29a and miR-29b, that are upregulated in muscle, adipose, and liver of diabetic animals. Overexpression of miR-29 in 3T3-L1 adipocytes suppresses insulinstimulated glucose uptake, an effect likely due to inhibition of protein kinase B/Akt activation. Consistent with its upregulation in diabetic animals, miR-29 expression is also increased in adipocytes cultured in high glucose and high insulin-containing media. This observation suggests a possible involvement of miR-29 in the development of insulin resistance; however, the mechanism behind the role of miR-29 in insulin resistance is not known. Although insulin-induced gene 1 (Insig1) and caveolin 2 (Cav2) were identified as miR-29 targets, their roles in insulin signaling and glucose metabolism remain to be clarified. Other miRNAs are also implicated in controlling the insulin receptor signaling pathway. However, the direct effects of these miRNAs on glucose metabolism remain to be established. For example, miR-145 can directly target and suppress the expression of insulin receptor substrate 1 (IRS1), a key component of the insulin receptor signaling pathway. Overexpression of miR-145 in colon cancer cells leads to growth arrest, a
193
phenotype reminiscent of IRS1 knockdown by siRNA in these cells. Another miRNA cluster, miR-183-96-182, targets multiple components of the insulin receptor signaling pathway, including IRS1, Rasa1, and Grb2. These observations suggest that for a given signaling pathway, multiple miRNAs can target different components of the signaling cascade, effectively shutting it off.
miRNA in the Treatment of Metabolic Diseases The implication of specific miRNAs in metabolic diseases (e.g., miR-29 in diabetes) has raised the hope that targeting these miRNAs may be a viable therapeutic approach to treat metabolic disorders. Various methods and tools are now available to modulate the expression of specific miRNAs in vivo. A gain of function can be achieved in vivo using viral-based methods to overexpress a synthetic form of any given miRNA in a desired tissue. A loss of function can be achieved in vivo by the delivery of antisense oligonucleotides specific to a target miRNA. In general, these antisense oligonucleotides are chemically modified to increase their stability and binding specificity. Several proof-of-principle studies have successfully demonstrated the feasibility and effectiveness of using adenovirus and lentivirus to deliver miRNA genes in mice. In addition to viral-based strategies, synthetic RNAs (e.g., antisense oligonucleotides) can be successfully delivered to tissues in vivo by conjugating synthetic RNAs to cholesterol and encapsulating them within the high- or low-density lipoproteins. However, this approach is limited because the encapsulated synthetic RNAs can be best delivered to only a few tissues such as the liver, intestine, and kidney. Perhaps a bigger problem with targeting miRNA to treat diseases lies in the specificity of the miRNA therapy. First, one miRNA can target several distinct genes and one gene can be targeted by multiple distinct miRNAs. It is not desirable to suppress those miRNAs that are not directly involved in disease pathogenesis. Thus, the potential off-target effects pose a significant challenge to miRNA-based therapy. Second, most of the miRNAs exhibit spatial, temporal, and tissue-specific expression patterns, characteristics necessary to their proper function. Therefore, a generic overexpression of miRNAs may not achieve the desired therapeutic effects. Whether miRNA-based therapeutic agents can be successfully deployed to treat metabolic diseases in clinical settings remains an open question.
Conclusion miRNAs comprise a diverse family of small noncoding RNAs that play important roles in fine-tuning gene expression and have been implicated in many fundamental physiologic processes. The roles of miRNA in metabolism (Table 1) are still being uncovered, and many miRNAs potentially important to this process are yet to be characterized. The immediate goals in the miRNA community are to validate the authenticity of the predicted miRNA genes in vivo and to clone new miRNA genes, establish their function, and identify their mRNA targets. While miRNA-based therapy is an attractive alternative to treat certain metabolic diseases, the questionable specificity of
194
Table 1
Metabolism Vitamins and Hormones | Roles of Micro-RNAs in Metabolism
The role of miRNAs in metabolism
miRNA
Metabolic regulation
Target tissues/cell lines
Target genes
miR375 miR9 miR-124a miR-14 miR-278 miR-143 miR-103 miR-200 miR-17-92 miR-27 miR-122 miR-29 miR-145 miR-183-96-182
Insulin secretion; a- and b-cell proliferation; b-cell development Insulin secretion Development and proliferation of b-cell Lipid metabolism in fat body Lipid metabolism in fat body; insulin resistance Adipogenesis Adipogenesis Adipogenesis Adipogenesis Adipogenesis Cholesterol and fatty acid metabolism Diabetes, insulin resistance Insulin signaling; growth Insulin signaling
Pancreas Pancreas Pancreas Fat body (Drosophila) Fat body (Drosophila) Human preadipocytes; 3T3-L1 cells 3T3-L1 Mouse ST2 cells 3T3-L1cells 3T3-L1 cells Liver Muscle, adipose, and liver Colon cancer cells
Mtpn, Usp1 OC2 Foxa2, Rab27A Unknown Expanded ERK5/MAPK7 Unknown Unknown Rb2/p130 PPAR-g and C/EBP-a HMG-CoA reductase Insig1 and Cav2 IRS1 IRS1, Rasa1, and Grb2
miRNA-based therapy and the challenge of delivering miRNA or anti-miRNA molecules to the desired target tissues in vivo will be the key issues that need to be addressed in the near future.
See also: Molecular Biology: MicroRNAs in Eukaryotes.
Further Reading Baroukh N, Ravier MA, Loder MK, et al. (2007) MicroRNA-124a regulates Foxa2 expression and intracellular signaling in pancreatic beta-cell lines. Journal of Biological Chemistry 282(27): 19575–19588. Bartel DP (2009) MicroRNAs: Target recognition and regulatory functions. Cell 136(2): 215–233. Esau C, Davis S, Murray SF, et al. (2006) miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metabolism 3(2): 87–98. Esau C, Kang X, Peralta E, et al. (2004) MicroRNA-143 regulates adipocyte differentiation. Journal of Biological Chemistry 279(50): 52361–52365. Gauthier BR and Wollheim CB (2006) MicroRNAs: ‘Ribo-regulators’ of glucose homeostasis. Nature Medicine 12(1): 36–38. He A, Zhu L, Gupta N, Chang Y, and Fang F (2007) Overexpression of mir-29, highly up-regulated in diabetic rats, leads to insulin resistance in 3T3-L1 adipocytes. Molecular Endocrinology 21(11): 2785–2794. Kru¨tzfeldt J, Rajewsky N, Braich R, et al. (2005) Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438(7068): 685–689.
Lin Q, Gao Z, Alarcon RM, Ye J, and Yun Z (2009) A role of miR-27 in the regulation of adipogenesis. FEBS Journal 276(8): 2348–2358. Lynn FC (2009) Meta-regulation: MicroRNA regulation of glucose and lipid metabolism. Trends in Endocrinology and Metabolism 20(9): 452–459. Pandey AK, Agarwal P, Kaur K, and Datta M (2009) MicroRNAs in diabetes: Tiny players in big disease. Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology 23(4–6): 221–232. Poy MN, Eliasson L, Krutzfeldt J, et al. (2004) A pancreatic islet-specific microRNA regulates insulin secretion. Nature 432(7014): 226–230. Poy MN, Hausser J, Trajkovski M, et al. (2009) miR-375 maintains normal pancreatic alpha- and beta-cell mass. Proceedings of the National Academy of Sciences of the United States of America 106(14): 5813–5818. Poy MN, Spranger M, and Stoffel M (2009) MicroRNAs and the regulation of glucose and lipid metabolism. Diabetes, Obesity and Metabolism 9(supplement 2): 67–73. Tang X, Tang G, and Ozcan S (2008) Role of microRNAs in diabetes. Biochimica et Biophysica Acta 1779(11): 697–701. Teleman AA, Maitra S, and Cohen SM (2006) Drosophila lacking microRNA miR-278 are defective in energy homeostasis. Genes and Development 20(4): 417–422. Wang Q, Li YC, Wang J, et al. (2008) miR-17-92 cluster accelerates adipocyte differentiation by negatively regulating tumor-suppressor Rb2/p130. Proceedings of the National Academy of Sciences of the United States of America 105(8): 2889–2894. Xie H, Lim B, and Lodish HF (2009) MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity. Diabetes 58(5): 1050–1057. Xie H, Sun L, and Lodish HF (2009) Targeting microRNAs in obesity. Expert Opinion on Therapeutic Targets 13(10): 1227–1238.