Endocrinology, doi:10.1210/en.2006-0147
Endocrinology Vol. 147, No. 6 2650-2656
Copyright © 2006 by The Endocrine Society
Minireview: Ribonucleic Acid Interference for the Identification of New Targets for the Treatment of Metabolic Diseases
Cristina M. Rondinone
Department Metabolic Diseases, Hoffmann-La Roche, Nutley, New Jersey 07110
Address all correspondence and requests for reprints to: Dr. Cristina M. Rondinone, Department Metabolic Diseases, Hoffmann-La Roche, 340 Kingsland Street, Nutley, New Jersey 07110. E-mail: cristina.rondinone{at}roche.com.
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Abstract
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Over the past years, RNA interference (RNAi) has exploded as a new approach to manipulate gene expression in mammalian systems. More recently, RNAi has acquired interest as a tool to identify new targets for therapeutic intervention. This review focuses on the current understanding of RNAi biology, how RNAi has been used to study the role of different genes in the pathogenesis of diabetes and obesity, and the use of RNAi screens for the identification of new targets for metabolic diseases. Also reviewed are the in vivo proof of principle experiments that provide the validation of these new targets for the development of RNAi-based therapeutics for diabetes.
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Introduction
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TARGET IDENTIFICATION is an essential first step in drug development that attempts to discover new targets, the modulation of which inhibits or reverses disease progression. One technology that has generated a lot of excitement is RNA interference (RNAi) (1). RNAi is a process in which long double-stranded RNA molecules (dsRNA) can induce sequence-specific silencing of gene expression in primitive and multicellular organisms (1). These long dsRNAs are processed by a ribonuclease called Dicer (2) into 21- to 23-nucleotide (nt) guide RNA duplexes termed short interfering RNA (siRNA) (3, 4, 5, 6, 7). The siRNA is subsequently used by an RNA-induced silencing complex (RISC), a protein-RNA effector nuclease complex that uses siRNA as a template to recognize and cleave RNA targets with similar nucleotide sequences. The composition of the RISC is not completely defined, but includes argonaute family proteins (8, 9). The RISC unwinds siRNAs and associates stably with the (antisense) strand that is complementary to target mRNA (10). Depending on the degree of homology between an siRNA and its target mRNA, siRNA-RISC complexes inhibit gene function by two distinct pathways (11). Most siRNAs pair imperfectly with their targets and silence gene expression by translational repression (12, 13, 14). This RNAi mechanism appears to operate most efficiently when multiple siRNA-binding sites are present in the 3'-untranslated region of the target mRNAs (15, 16). In some other cases, siRNAs exhibit perfect sequence identity with the target mRNA and inhibit gene function by triggering mRNA degradation (17).
Loss of function genetic screens using RNAi represent a powerful, unbiased approach for the identification of new drug targets by screening for target gene transcripts that, when silenced, affect the phenotype of interest (Fig. 1
). One of the issues of RNAi screens for target identification is the development of therapeutically relevant, cell-based assays underlying key intervention points for the pathology of different diseases. Thus, RNAi screens can be applied to numerous phenotypic cell-based assays in different cell types relevant to the individual disease. Subgenome libraries comprising a pathway, gene family, or gene set of interest focus on the biological relevance of high-priority genes. These can be genes identified from proteomics or microarray studies, genes commonly occurring in the disease state, or simply functionally related gene families. Genome-scale screens can reveal novel or unexpected pathways and gene families that can be disrupted to induce the phenotype and simultaneously provide a large background set of genes for determination of the importance of the biological impact. In addition to large-scale screening for target identification, RNAi has applications for target validation. The development of stable and inducible expression vectors driving the expression of short hairpin RNAs (shRNAs) has further expanded the application of RNAi in both tissue culture and animal models.
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FIG. 1. Target identification using RNA interference. Whole genome and subgenome siRNA libraries were used to develop siRNA screens in different organisms and cell types to identify new targets for metabolic diseases.
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This review focuses in the utilization of RNAi to elucidate different signaling pathways that are relevant to the understanding of the pathogenesis of metabolic diseases and to the discovery and validation of new targets for these diseases.
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Dissection of Insulin Signaling Using RNAi Technology
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One of the first attempts to use RNAi technology in metabolic diseases was to investigate the insulin signaling pathway. Insulin signaling is a required mechanism for normal glucose homoeostasis. The major metabolic pathways regulated by insulin include inhibition of gluconeogenesis in liver and stimulation of glucose transport in muscle and adipose tissue (18, 19). Insulin signaling also regulates diverse cellular functions, including apoptosis, mitogenesis, and membrane trafficking. The cellular effects of insulin are mediated by activation of the insulin receptor’s intrinsic tyrosine kinase activity that catalyzes phosphorylation of insulin receptor substrate proteins (IRS) (20), which recruit and activate phosphotidylinositol 3-kinase (PI3K), leading to conversion of phosphatidylinositol,4,5-diphosphate into phosphatidylinositol 3,4,5-trisphosphate and the activation of 3'-phosphoinositide-dependent kinase-1 (PDK1) and multiple protein kinases, including the atypical protein kinase C
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isoforms and Akt/protein kinase B (PKB) isoforms. Another pathway proceeds through Grb2/Sos and Ras, leading to activation of the MAPK isoforms ERK1 and ERK2 (18, 19, 20). There are still conflicting data related to the connection between pathways downstream of the insulin receptor leading to different insulin actions, such as mitogenesis, regulation of cell size, and glucose transport. Therefore, several investigators used RNAi technology to dissect these different pathways in Drosophila and mammalian cells.
RNAi in Drosophila cells
A recent report assessed the suitability of RNAi for dissecting signal transduction cascades in Drosophila using the well-characterized insulin signaling pathway (21). Similar to the mammalian insulin receptor, activation of the Drosophila insulin receptor triggers the activation of various intracellular effectors, including the MAPK and PI3K pathways (22, 23, 24, 25). Activation of the MAPK pathway by insulin results in the increased phosphorylation and activity of downstream effectors, including Downstream-of-Raf1 (DSOR1) (MAPK kinase), which subsequently phosphorylates and activates ERK-A.
As predicted, the lack of DSOR1 precludes the activation of ERK-A after insulin stimulation. In contrast, blocking ERK-A expression results in increased activation of DSOR1, confirming several reports reviewed by Schaeffer and Weber (26) that implicate ERK-A in down-regulation of the MAPK pathway.
RNAi applicability was also determined in another branch of the insulin signaling cascade (21). This branch of insulin receptor signaling involves phosphorylation of CHICO, the homolog of the vertebrate IRS family that activates PI3K leading to Drosophila v-akt murine thymoma viral oncogene homolog (DAKT)/PKB activation (27). S2 cells exposed to dsRNAs for CHICO were no longer able to activate DAKT/PKB in response to insulin. Conversely, cells treated with dsRNA corresponding to phosphatase and tensin homolog protein (PTEN), the negative regulator of this pathway (28), demonstrated an increase in DAKT/PKB activity with insulin treatment. These experiments highlighted the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome.
RNAi in mammalian cells
A recent report attempted to better understand the determinants of siRNA knockdown for use in high-throughput, cell-based screens (29). In this case, 148 siRNA duplexes targeting 30 genes within the PI3K pathway were selected and synthesized. A pathway-wide, cell-based, genetic screen was conducted to detect negative genetic regulators of PKB/Akt phosphorylation and activation. Interestingly, two known negative regulators of this phosphorylation, PTEN and PDK1 (28, 30, 31), were found. This proof of concept experiment helped to lay the foundation for genome-wide siRNA screens in mammalian cells and confirmed the observed roles of PTEN and PDK1 as negative regulators of PKB/Akt phosphorylation.
One of the challenging issues in the use of siRNA for target identification and validation is the transfection efficiency obtained in differentiated mammalian cells, such as adipocytes, muscle cells, hepatocytes, and ß-cells. Optimizations of these techniques are necessary to obtain good knockdown while maintaining the cells healthy and responsive to insulin. Czech’s group (32) was one of the first to develop a technique to transfect siRNA into adipocytes. The initial experiments showed that conditions developed for siRNA-mediated gene silencing in other cell types (33) worked well in 3T3-L1 fibroblasts, but failed to work in differentiated 3T3-L1 adipocytes. Therefore, they developed alternate methodology to transfect siRNA into 3T3-L1 adipocytes using electroporation (32). With this method, siRNA was introduced with virtually 100% efficiency into the cultured adipocytes with no detectable toxicity, and they dissected the insulin signaling pathway leading to glucose transport (Fig. 2) (34). RNAi-based depletion of components in the putative TC10 pathway [CAP, Cbl (Cas-BR-M murine ecotropic retroviral transforming sequence homolog)-activating protein; CrkII, V-CRK avian sarcoma virus CT10 oncogen homolog; and c-Cbl plus Cbl-b] or the phospholipase C
pathway failed to diminish glucose transport (34, 35). In contrast, this report showed that Akt2/PKBß was the key downstream intermediate within the PI3K pathway linked to insulin action on glucose transporter 4 (GLUT4) in cultured adipocytes, whereas PTEN was a potent negative regulator of this pathway.
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FIG. 2. Dissection of insulin signaling pathways involved in the regulation of insulin-stimulated glucose transport. Insulin receptor tyrosine kinase catalyzes the phosphorylation of IRS proteins that recruit and activate PI3K to form phosphatidylinositol (3,4,5) triphosphate, which leads to the activation of PDK-1. The protein kinase PDK1 can phosphorylate multiple downstream protein kinases, such as Akt/PKB and protein kinase C, resulting in the translocation of GLUT-4 to the plasma membrane. In a second proposed pathway, insulin receptor phosphorylates APS and c-Cbl proteins, and Cbl interacts with CAP, which leads to recruitment of CrkII/C3G and GTP binding to TC10, as shown. Foxo1, Forkhead box O1; SH2, Src homology 2 domain.
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Additional studies reported the application of siRNA- directed gene silencing to deplete both Akt1/PKB
and Akt2/PKBß in cultured 3T3-L1 adipocytes (32). Loss of Akt1 alone slightly impaired insulin-mediated glucose transport activity, but had no detectable effect on glycogen synthase kinase 3 (GSK-3) phosphorylation. In contrast, depletion of Akt2 alone by 70% inhibited approximately half the insulin responsiveness. Combined depletions of both kinases in these cells even more markedly attenuated insulin action on GLUT4 translocation, hexose transport activity, and GSK-3 phosphorylation. These data demonstrate a primary role of Akt2 in insulin signaling and significant functional redundancy of Akt1 and Akt2 isoforms in this pathway, and an absolute requirement of Akt protein kinases for regulation of glucose transport and GSK-3 in cultured adipocytes.
These results were confirmed by Katome et al. (36). Experiments with isoform-specific siRNA revealed that Akt2 and, to a lesser extent, Akt1 have essential roles in insulin-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis.
Similarly, a recent study (37) to determine the relative contributions of IRS-1 vs. IRS-2 in insulin signaling in L6 myotubes was performed using siRNA-mediated, specific gene silencing. The researchers concluded that insulin-stimulated Akt1 phosphorylation, actin remodeling, GLUT4 translocation, and glucose uptake were regulated mainly by IRS-1, whereas IRS-2 contributed selectively to ERK signaling. Thus, all these studies support the idea that RNAi is a useful technology to interrogate and dissect pathways relevant to metabolic diseases.
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Identification of Novel Targets for Metabolic Diseases
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Identification of new targets for metabolic diseases in Caenorhabditis elegans
The utility of RNAi-based gene silencing can also be extended to identify novel components that function in insulin signaling or other metabolic pathways and to discover new targets for therapeutic intervention. A reverse genetic screen of most of the C. elegans genome for defects in lipid storage has been completed by Gary Ruvkun’s laboratory (38). Using RNAi to disrupt the expression of each of the 16,757 worm genes, this group has systematically screened the C. elegans genome for genes necessary for normal fat storage. After addition of Nile red to Escherichia coli, the diet of C. elegans resulted in uptake and incorporation of the dye into lipid droplets in intestinal cells. Using this assay and the RNAi library, they screened for gene inactivation that affects fat content, fat droplet morphology, and the pattern of fat droplet deposition. They identified 305 gene inactivations that cause reduced body fat and 112 gene inactivations that cause increased fat storage. Some of the disruptions were in genes involved in the insulin and serotonin signaling pathways. Moreover, some of the genes in which inactivation caused reduced amounts of stored fat were already known to have a key role in mammalian fat or lipid metabolism, such as enzymatic components of the membrane lipid biosynthetic machinery, components of sterol metabolism, genes that function in the flux of glucose and glycerol energy metabolism, and genes involved in gastrointestinal digestion and uptake of food. Interestingly, several C. elegans genes that may function in food sensation and neuroendocrine signaling also resulted in aberrant fat content. This screen in C. elegans was the starting point to discover candidate genes involved in the pathogenesis of diabetes or obesity.
Recently, the same group reported a genome-wide functional genomic screen for longevity genes (39). They found that RNAi inactivation of 89 genes extended the C. elegans life span. Moreover, components of the daf-2/insulin-like signaling pathway were involved in longevity as were genes that regulate metabolism, signal transduction, protein turnover, and gene expression.
Identification of new targets for metabolic diseases using relevant mammalian cells
To identify gene products that regulate insulin signaling and metabolic pathways in cultured adipocytes, Czech’s laboratory has optimized the methods for transfection of adipocytes with siRNA such that 96-well plates can be used for functional assays in a relatively high throughput format (34). Interestingly, using this technology they were able to identify genes involved in glucose transport in 3T3-L1 adipocytes (40). One highly expressed gene that was found to regulate insulin-stimulated glucose transport in these cells was the transcriptional corepressor RIP140 (receptor-interacting protein-140), which was previously found to control fertility (40, 41). Remarkably, another laboratory independently reported that RIP140 knockout mice show enhanced energy expenditure and increased expression of uncoupling protein-1 in white fat cells (42). RIP140 seems also to be a major suppressor of adipocyte oxidative metabolism and mitochondrial biogenesis as well as a negative regulator of whole-body glucose tolerance and energy expenditure in mice (40), suggesting that RIP140 may be considered a candidate therapeutic target for the syndromes of obesity and type 2 diabetes.
Recently, a human RNAi library was used to elucidate signaling pathways leading to insulin resistance (43). Alterations in the early steps of insulin signaling, such as IRS-1 phosphorylation and degradation, have been recognized as important components of many insulin-resistant states (44). However, the basic mechanisms for the regulation of IRS-1 levels are not clear, but it appears that a rapamycin-dependent pathway triggers degradation through the proteasome pathway (45). To elucidate the signaling pathways involved in this pathway, an RNAi screen was used to identify kinases that prevent IRS-1 loss after chronic insulin treatment and enhance insulin-induced phosphorylation of Akt/PKB in human hepatoma cells. From approximately 500 kinases screened, 26 hits were identified and confirmed. Among those, mammalian target of rapamycin, protein kinase C
, c-Jun N-terminal kinase-1, and I-
-B-kinase 2 were found to be involved in the down-regulation of IRS-1 and insulin resistance in hepatocytes (43). In addition, a number of new kinases that were not known to be involved in insulin resistance or diabetes were identified. Thus, a mammalian target of rapamycin pathway as well as multiple kinase-dependent pathways were involved in the development of insulin resistance in hepatocytes.
A different approach used gene expression profiles in islets from 6-wk-old Zucker diabetic fatty rats and Zucker fatty rat controls to identify global changes in gene expression underlying ß-cell dysfunction (46). A total of 977 genes were found to be differentially regulated, comprising large groups of membrane and structural proteins, kinases, channels, receptors, transporters, growth factors, and transcription factors. Thus, a subset of those with no as yet defined role in the ß-cell was selected for additional study using siRNA. Interestingly, siRNA-mediated silencing of the Egr-1 gene inhibited proliferation of ß-cells in a glucose-independent manner, suggesting that reduced Egr-1 gene expression may contribute to decreased ß-cell proliferation and the consequent ß-cell failure observed in the later stages of type 2 diabetes.
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Validation of Targets in Vivo
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Insulin resistance is a major hallmark in the development of type 2 diabetes, which is characterized by an impaired ability of insulin to inhibit glucose output from the liver and to promote glucose uptake in muscle. Thus, the regulation of hepatic gluconeogenesis is an important process in the adjustment of the blood glucose level, and pathological changes in glucose production by the liver are a central characteristic in type 2 diabetes (18). Pharmacological intervention in signaling events that regulate the expression of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and the catalytic subunit glucose-6-phosphatase as well as glycogen synthesis and fatty acid oxidation in liver has been long regarded as a potential strategy for the treatment of metabolic aberrations associated with this disease. A number of studies used RNAi technology to target key genes involved in the regulation of gluconeogenesis and provided in vivo proof of principle for the development of RNAi-based therapeutics for diabetes. Some investigators have taken advantage of the relative tissue specificity of adenovirus for liver and the genetic specificity of shRNA-mediated RNAi to create liver-specific down-regulation of different genes. Kahns’s laboratory (47) developed an adenovirus-mediated RNAi technique that uses tail vein injection with shRNAs to substantially and stably knock down IRS-1 and IRS-2 expression specifically in the livers of mice to better understand the roles of IRS-1 and IRS-2 in hepatic insulin action. This group demonstrated that IRS-1 was more closely linked to the regulation of gluconeogenesis and Gck expression, whereas IRS-2 was involved in the regulation of lipogenesis. Moreover, the concomitant knockdown of IRS-1 and IRS-2 in liver resulted in fasting hyperglycemia, fasting hyperinsulinemia, insulin resistance, glucose intolerance, and dyslipidemia.
Another study showed the adenoviral delivery of peroxisome proliferator-activated coactivator-1 (PGC-1) siRNA to the liver (48). PGC-1 is a nuclear hormone coactivator whose hepatic expression is elevated in mouse models of this disease, where it promotes constitutive activation of gluconeogenesis and fatty acid oxidation (49). PGC-1-deficient mice, generated by adenoviral delivery of PGC-1 RNAi to the liver, experienced fasting hypoglycemia and enhanced hepatic insulin sensitivity with reduced expression of the mammalian Tribbles homolog-3, a fasting-inducible inhibitor of the serine-threonine kinase Akt/PKB (50). These results indicate a link between nuclear hormone receptor and insulin signaling pathways and suggest a potential role for Tribbles homolog-3 inhibitors in the treatment of type 2 diabetes. Thus, adenovirus-mediated RNA interference represents a promising technique to further understand the complex network of hepatic gene function without the pleiotropic side effects that could occur in knockout animals.
A third group designed a therapeutic, vector-based RNAi approach to induce posttranscriptional gene silencing of hepatic PEPCK, the rate-controlling enzyme in gluconeogenesis (51). Using nonviral gene delivery, PEPCK partial silencing in liver was sufficient to induce lowered blood glucose, improved glucose tolerance, and decreased circulating free fatty acids and triglycerides in treated animals. These data validate liver-specific intervention at the level of PEPCK for diabetes gene therapy.
A different approach to target liver was through the administration of chemically modified siRNAs that resulted in silencing of apolipoprotein B (apoB) mRNA in liver and jejunum and decreasing plasma levels of apoB protein (52). ApoB is a molecule involved in the metabolism of cholesterol; the concentrations of this protein in human blood samples correlate with those of cholesterol. Higher levels of both compounds are associated with an increased risk of coronary heart disease (53, 54). Intravenous injections of the siRNA-cholesterol conjugates in mice resulted in a lowering of the levels of blood cholesterol comparable to that in mice in which the apoB gene had been deleted (55). These results demonstrate that siRNA can be delivered systemically to target the liver and suggest that RNAi has the potential to become a new therapeutic for the treatment of metabolic diseases.
Chemically synthesized siRNAs were also tested for their in vivo gene knockdown ability in the brain. In the first such attempt, siRNA directed against the gene expressing agouti-related protein (AGRP) was locally injected into the hypothalamic arcuate nucleus of adult mice (56). Decreased endogenous hypothalamic AGRP levels in the central nervous system resulted in an increase in the metabolic rate and a decrease in body weight. These results are in agreement with the observation that transgenic overexpression of the AGRP gene leads to hyperphagia and obesity (57) and injections of synthetic analogs of AGRP and of AGRP itself also stimulate food intake and body weight (58). Thus, these data suggest that antagonism of AGRP may reduce food intake and body weight, potentially serving as a therapy for obesity.
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Current Limitations and Future Challenges
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One of the potential advantages of RNAi technology is the ability to design precisely targeted knockdown for almost any gene. Specificity is one of the greatest advantages offered by RNAi. Indeed, the initial description of these siRNAs demonstrated that 21-nucleotide siRNA duplexes specifically suppress the expression of endogenous and heterologous genes in different mammalian cell lines (33). However, off-target effects are still a potential problem. These off-target effects have been correlated with the concentration of siRNAs (59) as well as similarities between the off-target transcripts and the 5' ends of siRNAs (60, 61). Another issue is the nonspecific toxicity caused by both the delivery vehicle and the siRNA itself that may induce or potentiate unexpected cellular responses, such as interferon and immune responses (62, 63). In addition, the nature of siRNA effectors themselves may trigger the induction of cellular defense mechanisms. Consequently, careful design and screening of each candidate siRNA and delivery vehicle will be necessary to identify and minimize all potential adverse effects.
One of the major challenges for using the RNAi platform in vivo is delivery. Optimizing systemic delivery requires stabilization of the siRNA, targeting to the correct tissue, and facilitation of cellular uptake. Advances to improve stability and cellular uptake of siRNA include direct chemical alteration of the nucleic acid (52) and packaging the siRNAs into protective particles (52, 64, 65, 66, 67). To target the siRNA to particular cell types, different ligands and antibodies are being conjugated to the siRNA (68, 69). To obtain efficient and long-lived gene silencing using RNAi in cells and tissues, many groups have developed a variety of viral vectors to deliver siRNAs both in vitro and in vivo (47, 48), but continued work is needed to achieve the efficiency required. Discoveries in the field of RNAi biochemistry coupled with technological breakthroughs will permit the creation in the near future of effective RNAi reagents to optimize this technology for applications in animal studies and clinical trials.
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Conclusion
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RNAi has rapidly emerged as a tool to interrogate the function of candidate genes and, after the creation of siRNA libraries, has permitted the advance of genetic analysis of normal physiological and disease processes. RNAi technology is facilitating the dissection of signaling pathways involved in the development of obesity, insulin resistance, and diabetes and the identification and validation of novel targets for therapeutic intervention. RNAi can be used to silence endogenous genes involved in the cause or pathway of metabolic diseases and holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called nondrugable targets.
The development of stable and inducible expression vectors driving the expression of shRNA has further expanded the application of RNAi in both tissue culture and animal models. Furthermore, several studies have demonstrated efficient in vivo delivery of siRNAs and therapeutic benefit in diabetic and obese mice. It is hoped that ongoing and future preclinical studies in animal models will help optimize RNAi therapeutics for applications in humans. Although the development of RNAi-based therapeutics for diabetes is in its infancy, early clinical studies are soon to begin assessing the use of this new class of therapeutics in tackling metabolic diseases, including diabetes and obesity.
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Acknowledgments
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Apologies are extended to colleagues whose work could not be cited due to space limitations.
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Footnotes
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C.M.R. is employed by Hoffmann-La Roche, Inc.
First Published Online March 23, 2006
Abbreviations: AGRP, Agouti-related protein; apoB, apolipoprotein B; DKAT, Drosophila v-akt murine thymoma viral oncogene homolog; DSOR-1, Downstream-of-Raf1; dsRNA, double-stranded RNA; GLUT, glucose transporter; GSK-3, glycogen synthase kinase 3; IRS, insulin receptor substrate protein; nt, nucleotide; PDK1, 3'-phosphoinositide-dependent kinase-1; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1, peroxisome proliferator-activated- coactivator-1; PI3K, RNA-induced silencing complex; PKB, protein kinase B; PTEN, phosphatase and tensin homolog protein; RIP140, receptor-interacting protein-140; RISC, RNA-induced silencing complex; RNAi, RNA interference; shRNA, short hairpin RNA; siRNA, short interfering RNA.
Received February 6, 2006.
Accepted for publication March 7, 2006.
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Abstract
Introduction
