miR-33a/b contribute to the regulation of fatty acidmetabolism and insulin signalingAlberto Dávalosa,1, Leigh Goedekea,1, Peter Smibertb, Cristina M. Ramíreza, Nikhil P. Warriera, Ursula Andreoa,Daniel Cirera-Salinasa,c,d, Katey Raynera, Uthra Sureshe, José Carlos Pastor-Parejaf, Enric Espluguesc,d,g, Edward A. Fishera,Luiz O. F. Penalvae, Kathryn J. Moorea, Yajaira Suáreza, Eric C. Laib, and Carlos Fernández-Hernandoa,2

aDepartments of Medicine and Cell Biology, Leon H. Charney Division of Cardiology and the Marc and Ruti Bell Vascular Biology and Disease Program,New York University School of Medicine, New York, NY 10016; bDepartment of Developmental Biology, Sloan–Kettering Institute, New York, NY 10065;cGerman Rheumatism Research Center (DRFZ), A. Leibniz Institute, 10117 Berlin, Germany; dCluster of Excellence NeuroCure, Charite-Universitatsmedizin,10117 Berlin, Germany; eChildren’s Cancer Research Institute, University of Texas Health Science Center, San Antonio, TX 78229; fDepartment of Genetics,Yale University School of Medicine, New Haven, CT 06519; and gDepartment of Immunobiology, Yale University School of Medicine, New Haven, CT 06520

Edited by Joseph L. Witztum, University of California at San Diego, La Jolla, CA, and accepted by the Editorial Board April 22, 2011 (received for reviewFebruary 9, 2011)

Cellular imbalances of cholesterol and fatty acid metabolism resultin pathological processes, including atherosclerosis and metabolicsyndrome. Recent work from our group and others has shownthat the intronic microRNAs hsa-miR-33a and hsa-miR-33b are lo-cated within the sterol regulatory element-binding protein-2 and-1 genes, respectively, and regulate cholesterol homeostasis inconcert with their host genes. Here, we show that miR-33a and-b also regulate genes involved in fatty acid metabolism and in-sulin signaling. miR-33a and -b target key enzymes involved inthe regulation of fatty acid oxidation, including carnitine O-octa-niltransferase, carnitine palmitoyltransferase 1A, hydroxyacyl-CoA-dehydrogenase, Sirtuin 6 (SIRT6), and AMP kinase subunit-α. More-over, miR-33a and -b also target the insulin receptor substrate 2,an essential component of the insulin-signaling pathway in theliver. Overexpression of miR-33a and -b reduces both fatty acidoxidation and insulin signaling in hepatic cell lines, whereas in-hibition of endogenous miR-33a and -b increases these two met-abolic pathways. Together, these data establish that miR-33a and-b regulate pathways controlling three of the risk factors of met-abolic syndrome, namely levels of HDL, triglycerides, and insulinsignaling, and suggest that inhibitors of miR-33a and -b may beuseful in the treatment of this growing health concern.

lipid homeostasis | posttranscriptional regulation | cardiovascular disease

Many diseases result from perturbations in lipid homeostasis,including atherosclerosis, type II diabetes, and metabolic

syndrome (1–4). The intracellular and membrane levels of fattyacids and cholesterol are under constant surveillance and arecoordinated with de novo lipid biosynthesis by endoplasmic re-ticulum (ER)-bound sterol regulatory element-binding proteins(SREBPs) (5–7). The SREBP family of basic helix–loop–helix–leucine zipper (bHLH-LZ) transcription factors consists ofSREBP-1a, SREBP-1c, and SREBP-2 proteins that are encodedby two unique genes, Srebp-1 and Srebp-2 (5–7). The SREBPsdiffer in their tissue-specific expression, their target gene selec-tivity, and the relative potencies of their trans-activation domains.SREBP-1c regulates the transcription of genes involved in fattyacid metabolism, such as fatty acid synthase (FASN) (5–7).SREBP-2 regulates the transcription of cholesterol-related genes,such as 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),which catalyzes a rate-limiting step in cholesterol biosynthesis,and the low-density lipoprotein receptor (LDLr), which importscholesterol from the blood (5–7). Increased SREBP activitycauses cholesterol and fatty acid accumulation and down-regu-lates the SCAP/SREBP pathway by feedback inhibition. In thisway, lipid metabolism within cells is tightly regulated.In addition to classical transcriptional regulators, a class of

noncoding RNAs, termed microRNAs (miRNAs), has emergedas critical regulators of gene expression acting predominantly atthe posttranscriptional level (8–10). These short (22 nt) double-

stranded regulatory noncoding RNAs are encoded in the ge-nome, and most are processed from primary transcripts by thesequential actions of Drosha and Dicer enzymes (8–10). In thecytoplasm, mature miRNAs are incorporated into the cytoplas-mic RNA-induced silencing complex (RISC) and bind to par-tially complementary target sites in the 3′ UTRs of mRNA.miRNA targeting of mRNAs inhibits their expression throughmRNA destabilization, repression of translation, or a combina-tion of both processes (8–10).We and others provided identification of a highly conserved

miRNA family, miR-33, within the intronic sequences of theSrebp genes in organisms ranging from Drosophila to humans(11–14). Two miR-33 genes are present in humans: miR-33b,which is present in intron 17 of the Srebp-1 gene on chromosome17, andmiR-33a, which is located in intron 16 of the Srebp-2 geneon chromosome 22. In mice, however, there is only one miR-33gene, which is conserved with human miR-33a and located withinintron 15 of the mouse Srebp-2 gene.We recently showed that miR-33a is cotranscribed with its host

gene Srebp-2 like many intronic miRNAs, and it targets genes in-volved in cholesterol export, including the adenosine triphosphatebinding cassette (ABC) transporters ABCA1 and ABCG1 and theendolysosomal transport protein Niemann-Pick C1 (NPC1) (14).This regulatory function of miR-33a ensures that the cell is pro-tected under low sterol conditions from additional sterol loss. Inaddition to this role in maintaining cholesterol homeostasis,we now show that miR-33a and -b also regulate fatty acid metab-olism and insulin signaling. We identify putative binding sites formiR-33 in the 3′ UTR of carnitine O-octaniltransferase (CROT),carnitine palmitoyltransferase 1A (CPT1a), hydroxyacyl-CoA-de-hydrogenase (HADHB), AMP kinase subunit-α (AMPKα), andinsulin receptor substrate 2 (IRS2) and show that miR-33a and -bspecifically inhibit the expression of these genes. The physiologicalrelevance of this targeting is revealed by miR-33 overexpression inhepatic cells, which reduces both fatty acid oxidation and insulinsignaling. Furthermore, inhibition of endogenous miR-33 increa-ses the expression of CROT,CPT1a, HADHB,AMPKα, and IRS2and up-regulates fatty acid oxidation and insulin signaling. To-

Author contributions: C.F.-H. designed research; A.D., L.G., P.S., C.M.R., N.P.W., U.A.,D.C.-S., U.S., L.O.F.P., Y.S., and C.F.-H. performed research; J.C.P.-P. and E.C.L. contributednew reagents/analytic tools; A.D., L.G., P.S., C.M.R., U.A., K.R., U.S., J.C.P.-P., E.E., E.A.F.,L.O.F.P., K.J.M., Y.S., E.C.L., and C.F.-H. analyzed data; and L.G. and C.F.-H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. J.L.W. is a guest editor invited by the EditorialBoard.1A.D. and L.G. contributed equally to this work.2To whom correspondence should be addressed. E-mail: carlos.fernandez-hernando@nyumc.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1102281108/-/DCSupplemental.

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gether, these data suggest that feedback loops involving SREBPsand miR-33a and -b balance cholesterol metabolism, fatty acidoxidation, and insulin signaling, three of the major risk factors ofmetabolic syndrome (1, 3, 15).

ResultsmiR-33 Targets Genes Regulating β-Oxidation of Fatty Acid andInsulin Signaling. We have previously described the presence ofmiR-33a in the Srebp-2 gene. miR-33a is found within the sameintron of Srebp-2 from many animal species, including large andsmall mammals, chickens, and frogs. Interestingly, the fruit fly D.melanogaster also has a highly conserved mature form ofmiR-33a,but these organisms do not synthesize sterols. SREBP in fliesregulates fatty acid metabolism (16), which is reminiscent of thefunction of the Srebp-1 gene in mammals (6). As shown in Fig. S1A and B, miR-33b is synchronously expressed with SREBP-1c inhuman hepatic Huh7 cells treated with an agonist of the liver Xreceptor (LXR), a transcriptional regulator of Srebp-1c expres-sion. Kinetic analysis of miR-33b induction revealed a concomi-tant increase in miR-33b and SREBP-1c expression, consistentwith their coregulation. Thus, we postulated that miR-33a and -b,which differ only in 2 nt (Fig. S1C), might, therefore, regulategenes involved in lipid metabolism. To search for potential tar-gets, we performed gene ontology and biological associationanalyses using Pathway Studio 7 software (Ariadne Genomics)and looked for enrichment of specific target genes associated withlipid metabolism. As shown in Fig. S2 (red box), several genesinvolved in fatty acid β-oxidation have predicted targets for miR-33, including CROT, CPT1a, HADHB and AMPKα (17–20).CROT, a carnitine acyltransferase that catalyzes the reversibletransfer of fatty acyl between CoA and carnitine, provides a cru-cial step in the transport of medium and long-chain acyl-CoA out

of the mammalian peroxisome to the cytosol and mitochondria(17–20). CPT1a is a mitochondrial enzyme that mediates thetransport of long fatty acids across the membrane by binding themto carnitine, and it is the rate-limiting enzyme that regulates fattyacid oxidation (17–20). HADHB is the β-subunit of the mito-chondrial trifunctional protein, which catalyzes the last three stepsof mitochondrial β-oxidation of long-chain fatty acids, whereasAMPKα stimulates hepatic fatty acid oxidation and ketogenesis(17–20). Interestingly, we also identified IRS2, a component of theinsulin-signaling pathway, as a potential target of miR-33.To determine whether miR-33b targets these predicted target

genes, we generated reporter constructs with the luciferasecoding sequence fused to the 3′ UTRs of these genes. miR-33bmarkedly repressed the activity of the Crot, Cpt1a, Hadhb,Ampkα, and Irs2 3′ UTR luciferase constructs (Fig. S3). Fur-thermore, mutation of the miR-33 target sites in these constructsrelieved miR-33b repression of the 3′ UTR of Crot, Cpt1a,Hadhb, Ampkα, and Irs2, consistent with a direct interaction ofmiR-33b with these sites (Fig. S3). Mutation of both miR-33 sitesin the 3′ UTR of Crot was necessary to completely reverse theinhibitory effects of miR-33 (Fig. S3).We next determined the effect of miR-33 onmRNA and protein

expression of CROT, CPT1a, HADHB, AMPKα, IRS2, and lipid-related genes that lack predictedmiR-33 binding sites. Transfectionof Huh7 cells with miR-33b (32-fold increase expression) signifi-cantly inhibited the mRNA levels of CROT, CPT1a, HADHB,AMPKα, and IRS2 (Fig. 1A). Notably, inhibition of endogenousmiR-33b using anti–miR-33b oligonucleotides (threefold decreaseexpression) increased the mRNA expression of CROT, CPT1a,AMPKα, and IRS2 in Huh7 cells (Fig. 1B), consistent with a physi-ological role for miR-33b in regulating the expression of thesegenes. Similar regulation of these genes by miR-33b was also seen

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Fig. 1. Posttranscriptional regulation of IRS2, AMPKα, CROT, CPT1a, and HADHB by miR-33b. Quantitative RT-PCR expression profile of selected miR-33predicted target and other related genes in human hepatic Huh7 cell line (A) after overexpressing miR-33b and (B) after endogenous inhibition of miR-33b byanti–miR-33b. Western blot analysis of HepG2 cells (C) overexpressing or (D) inhibiting endogenous miR-33b. Heat shock protein (HSP)90 bands are theloading control. (E) Specificity of miR-33b on fatty acid metabolism-related genes. qRT-PCR array analysis of fatty acid metabolism-related genes from HepG2cells transfected with Con miR or miR-33. Data are the mean ± SEM and are representative of more than or equal to three experiments. *P ≤ 0.05.

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at the protein level (Fig. 1C andD).We next investigated the effectof manipulating miR-33 levels in vivo in mice using lentivirusesencoding premiR-33, anti–miR-33, or a control. Efficient lentiviraldelivery was previously confirmed (14). Consistent with our in vitroresults, miR-33 reduced hepatic CROT, HADHB, CPT1a, IRS2,and ABCA1 mRNA expression (Fig. S4). Conversely, miceexpressing anti–miR-33 showed a modest increase of CROT,CPT1a, IRS2, and ABCA1 mRNA expression, although the effectwas not statistically significant (Fig. S4).To show the specificity of miR-33b targeting of CROT, CPT1a,

HADHB, AMPKα, and IRS2, we assessed the effect of miR-33boverexpression in HepG2 cells on other fatty acid metabolism-related genes using an array that included 84 genes involved infatty acid transport and biosynthesis, ketogenesis, and ketone bodymetabolism. Whereas CROT, CPT1a, and AMPKα were pre-dictably down-regulated by miR-33, we did not observe changes inthe expression of non–miR-33 targets (Fig. 1E). Furthermore,other genes containing putative miR-33 binding sites such as cit-rate synthase (CS) and HMGCR were not affected either at themRNA level (Fig. S5A) or by 3′ UTR activity (Fig. S5B).

miR-33a and miR-33b Have Similar Targeting Effects on GenesRegulating Cholesterol Metabolism, β-Oxidation of Fatty Acids, andInsulin Signaling. miR-33a and -b have identical seed sequences

but differ in 2 of 19 nt of the mature RNA (Fig. S1C). To de-termine whether miR-33a and -b have similar effects on CROT,CPT1a, HADHB, AMPKα, Sirtuin 6 (SIRT6), and IRS2 proteinexpression, we transfected Huh7 cells with a control miR, miR-33a, or -b. As seen in Fig. S6, miR-33a and -b inhibited CROT,CPT1a, HADHB, AMPKα, SIRT6, and IRS2 protein expressionto a similar extent. In addition, both miR-33a and -b significantlyinhibited the 3′ UTR activity of Crot, Cpt1a, Hadhb, Ampkα, andIrs2 with only modest differences, indicating that the 2-nt vari-ation in the mature forms of miR-33a and -b does not appre-ciably affect the gene targeting by these miRNAs.

miR-33 Inhibits Cellular Fatty Acid Oxidation. To evaluate the effectsof miR-33a and -b on fatty acid β-oxidation, we measured therelease of [14C]-carbon dioxide from the oxidation of [14C]-oleate.miR-33b overexpression (27-fold increase) markedly reduced thefatty acid β-oxidation in Huh7 (Fig. 2A) cells. Conversely, in-hibition of endogenous miR-33b expression using anti–miR-33(2.6-fold increase) increased the rate of fatty acid β-oxidation(Fig. 2B). We next evaluated the accumulation of neutral lipidsand lipid droplet formation in Huh7 cells incubated with oleatefor 24 h and then starved for the next 24 h. In agreement with thereduced fatty acid β-oxidation rates observed in hepatic cellsoverexpressing miR-33b, Huh7 cells transfected with miR-33b

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Fig. 2. miR-33b regulates human hepatic β-oxidation and lipid homeostasis in Drosophila. (A and B) Relative rate of β-oxidation from Huh7 cells transfectedwith miR-33 (A) and anti–miR-33b (B). (C) Analysis of neutral lipid accumulation of Huh7 cells transfected with Con-miR or miR-33b and stained with Bodipy(green) and DAPI (blue). (D) Analysis of triglyceride content of Huh7 cells transfected with miR-33b at 0 and 24 h of starvation. (E) Analysis of triglyceridesynthesis of Huh7 cells transfected with Con-miR or miR-33 and stimulated or not stimulated with insulin. (F) Northern blot analysis of miR-33, miR-8, and2S rRNA of transgenic Drosophila overexpressing miR-33 or control transgene in the fat body [genotype: Cg-gal4, upstream activating sequence (UAS)-myrRFP, and UAS-transgene; abbreviated as Cg > transgene]. miR-8 is used as the control. (G) Neutral lipid accumulation in the fat body of Cg > DsRed and Cg> DsRed-miR-33 stained with Bodipy (green), Hoechst 33352 (blue), and the transgene (red). (Scale bar: 30 μm.) (H) Analysis of triglyceride content oftransgenic Drosophila overexpressing miR-33 or control transgene in the fat body before and after starvation.

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accumulated more triglycerides (TAG) in larger lipid droplets(Fig. 2 C and D). The increase in triglyceride content was in-dependent of changes in triglyceride synthesis rates. As seen inFig. 2E, miR-33 expression did not alter basal and insulin-in-duced triglyceride synthesis.Because CPT1a is also a target of miR-33 in Drosophila, we set

out to determine if miR-33 plays a role in maintaining lipid ho-meostasis in Drosophila. To do this, we generated a transgenic flythat overexpressed miR-33 in the fat body (Fig. 2F). We hypoth-esized that miR-33–overexpressing flies would retain TAG andfatty acids upon starvation as a consequence of reduced fatty acidoxidation, which should manifest as an increase in lipid storage.Accordingly, fat bodies from starved miR-33 transgenic flies, dis-sected and stained with Bodipy, showed large lipid droplets inmany cells compared with control flies (Fig. 2G). Similarly, fliesoverexpressing miR-33 accumulated more TAG upon starvation(Fig. 2H), indicating that the function of miR-33 in regulating fattyacid oxidation is conserved from Drosophila to humans.

miR-33 Regulates Insulin Signaling. To further explore our obser-vation that miR-33 inhibits IRS2 expression, we next assessed theeffect of miR-33 on insulin signaling. IRS2 is a cytoplasmic sig-naling molecule that mediates the effects of insulin, insulin-likegrowth factor 1, and other cytokines by acting as a molecularadaptor between receptor tyrosine kinases and downstreameffectors (21–23). To test the role of miR-33 in regulating insulin

signaling, we analyzed the effect of miR-33 overexpression ontwo of the main downstream effectors of IRS2: the PI3K/AKTand rat sarcoma (RAS)/RAF/ERK pathways (21–23). As seen inFig. 3 A and B, Huh7 cells transfected with miR-33b showedreduced AKT and ERK phosphorylation after insulin stimulation,indicative of reduced IRS2 function. Similar results were ob-served when we analyzed the AKT activation using an in vitrokinase assay (Fig. 3C). To determine whether or not IRS2 over-expression rescues the miR-33 overexpression effect on AKTphosphorylation, we transfected Huh7 cells with IRS2 cDNA thatlacked the 3′ UTR sequence. As seen in Fig. 3 D and E, IRS2expression rescued the AKT activation on insulin stimulation inmiR-33b–overexpressing cells.To gain insights into the role of miR-33 in regulating insulin

signaling, we assessed the effect of miR-33b on 2-deoxyglucoseuptake after insulin stimulation. As seen in Fig. 3F, miR-33boverexpression reduced insulin-induced 2-deoxyglucose (2-DOG)uptake in Huh7 cells. We also assessed FoxO1 cellular local-ization in insulin-stimulated cells. FoxO1 is a well-defined targetdownstream of the conserved insulin/target of rapamycin (TOR)signaling network that has important roles in the regulation ofprocesses as diverse as cellular growth, stress resistance, and en-ergy homeostasis (24). To date, several proteins are known tointeract with FoxO transcription factors, regulating their in-tracellular localization and/or activity. One of the best docu-mented is the AKT/protein kinase B (PKB) kinase, which

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Fig. 3. miR-33b regulates insulin signaling. (A and B) miR-33b impairs insulin signaling by reducing AKT phosphorylation in hepatic Huh7 cells. (C) In vitro AKTkinase assayof postinsulin-stimulated and immunoprecipitated total AKT fromHuh7 cells. GSK-3 fusion proteinwas used as a substrate and assayed for phosphor-GSK-3α/β (Ser-219). (D and E) IRS2 overexpression rescues Akt phosphorylation in miR-33b–transfected cells after insulin stimulation. (F) miR-33b overexpressionreduces 2-deoxyglucose (2-DOG) uptake inHuh7 cells treatedwith insulin. (G) Quantitative RT-PCR array analysis of insulin signaling relatedgenes fromHuh7 cellstransfected with Con miR or miR-33. Data are the mean ± SEM and are representative of more than or equal to three experiments. *P ≤ 0.05.

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phosphorylates FoxO in three conserved sites, leading to FoxOcytoplasmatic retention and transcriptional inactivation. To testthe effect of miR-33 on FoxO1 localization, we transfected Huh7with a con-miR or miR-33b and transduced the cells witha FoxO1-GFP adenovirus. FoxO1-GFP localized primarily to thenucleus in starved cells transfected with Con-miR (Fig. S7A Up-per) or miR-33b (Fig. S7B Upper). Treatment with insulin inducedFoxO1-GFP translocation from the nucleus to the cytoplasm inCon-miR–transfected cells (Fig. S7A Lower), whereas cellstransfected with miR-33b had the cellular distribution reversed(Fig. S7B Lower). Interestingly, IRS2 overexpression rescued theeffect of miR-33 overexpression on FoxO1 intracellular localiza-tion (Fig. S7 C and D). Together, these experiments identify miR-33 as an important regulator of the insulin-signaling pathways.In addition, we also assessed the effect of miR-33b over-

expression in Huh7 cells on other insulin-related genes using anarray that included 84 genes involved in carbohydrate and lipidmetabolism and target genes for insulin signaling. As expected,IRS2 was down-regulated in Huh7 cells transfected with miR-33b(Fig. 3D). Moreover, glucokinase (GCK), fibroblast growth factorreceptor substrate 2 (FRS2), acetyl-CoA carboxylase-α (ACACA),and peroxisome proliferator-activated–γ (PPARG) were alsoinhibited (Fig. 3D). Interestingly, FRS2 is also a predicted targetof miR-33a and -b. FRS2 has been suggested to participate ininsulin signaling by recruiting Src-homology-phosphatase 2 (SHP2)and hence, could function as a docking molecule similar to insulinreceptor substrate proteins.In addition to IRS2 and FRS2, our bioinformatic analysis iden-

tified a third miR-33 predicted target involved in glucose homeo-stasis: the histone deacetylase SIRT6 (25, 26). SIRT6-deficientmice develop normally but succumb to lethal hypoglycemia early inlife, suggesting an important role of SIRT6 in regulating glucosemetabolism (25, 26). Interestingly, it has also been recently repor-ted that hepatic-specific disruption of SIRT6 inmice results in fattyliver formation because of enhanced glycolysis and triglyceridesynthesis (27). To confirm that miR-33b targets SIRT6, we clonedthe Sirt6 3′ UTR into a luciferase reporter construct. miR-33bmarkedly repressed the 3′ UTR activity of Sirt6 (Fig. S8A), andmutation of themiR-33 target sites in the 3′UTR relievedmiR-33brepression of Sirt6, consistent with a direct interaction of miR-33bwith these sites (Fig. S8A). Furthermore, miR-33b overexpressionsignificantly inhibited the SIRT6mRNAand protein levels inHuh7cells (Fig. S8 B and C), whereas inhibition of endogenous miR-33bby anti–miR-33 increased the expression of SIRT6 (Fig. S8 B andC). Although these data are consistent with a physiological role formiR-33b in regulating SIRT6 expression, this is unlikely to con-tribute to the regulation of fatty acid metabolism by miR-33b, be-cause inhibition of SIRT6 expression by siRNA only modestlydecreased fatty acid β-oxidation (Fig. S8D).Additional experiments, including RIPseq and proteomics, are

warranted to understand the miR-33 regulatory network and itsimplication in lipid and carbohydrate metabolism.

DiscussionWe and others have recently established that, during sterol-limited states, miR-33a is coincidentally generated with Srebp-2transcription and works to increase cellular cholesterol levels bylimiting cholesterol export through the down-regulation of ABCtransporters, ABCA1 and ABCG1 (12–14). Importantly, thesepathways regulate circulating HDL levels through their roles inHDL biogenesis and cellular cholesterol efflux (12–14). We nowshow that a second member of the miR-33 family, miR-33b, iscoregulated with the human Srebp1 gene and targets genes in-volved in fatty acid oxidation and insulin signaling. Together,these results suggest a paradigm in which miR-33a and miR-33bact in concert with their host genes, Srebp-2 and Srebp-1, to boostintracellular cholesterol and fatty acid levels by balancing tran-scriptional induction and posttranscriptional repression of lipid

metabolism genes. Notably, we show that miR-33a and miR-33bhave overlapping gene targets, suggesting that cotranscription ofSrebp-2 and miR-33a or Srebp-1 and miR-33b would be predictedto regulate both cholesterol metabolism and fatty acid oxidation;this establishes a model of reciprocal regulation of cholesteroland fatty acid metabolism by SREBPs (Fig. 4).The presence of miR-33a in the intron of Srebp-2 is remarkably

conserved in many species, including the fruit fly D. melanogaster.This was notable, because Drosophila neither synthesizes sterolsnor expresses ABCA1. Interestingly, the Srebp gene of Drosophilacontrols fatty acid production, and our bioinformatic analysis ofmiR-33a and -b target genes revealed an enrichment of genesinvolved in fatty acid metabolism. We identify herein six miR-33target genes that regulate fatty acid metabolism and insulin sig-naling, including CPT1a, CROT, HADHB, AMPKα, SIRT6, andIRS2. Importantly, we show that miR-33 overexpression reducesfatty acid oxidation in hepatic cell lines. While this manuscriptwas in preparation, Gerin et al. (11) reported similar effects ofmiR-33 on CROT, CPT1a, and HADHB expression. Finally, weshow that miR-33 transgenic flies show increased lipid accumu-lation in tissues, suggesting that miR-33 regulation of fatty acidmetabolism pathways is evolutionarily conserved.Our work also identifies miR-33a and -b as regulators of insulin

signaling. By inhibiting expression of IRS2 in hepatic cells, miR-33a and -b reduce the activation of downstream insulin signalingpathways, including AKT and ERK. Although our previous workestablished that miR-33a and Srebp-2 are cotranscribed duringstates of cholesterol depletion (14), the changes in Srebp-2 andmiR-33a transcription were quite small in this setting. By contrast,Srebp-1c is transcribed at extremely high levels in response to in-sulin (6, 28), which may be particularly relevant in the setting ofmetabolic syndrome, where insulin resistance is accompanied byincreased triglycerides and plasma levels of very low density lipo-protein (VLDL) as well as reduced HDL levels (1, 3). Our dataindicate that miR-33a and -b impact pathways influencing three ofthe primary risk factors in this disease, namely insulin resistance,

Fig. 4. Potential role of SREBPs and miR-33a and -b in metabolic syndrome.In hepatocytes, conditions of low intracellular cholesterol (or statins) induceSREBP-2, leading to increased lipoprotein uptake and endogenous cholesterolbiosynthesis. Hyperinsulinemia or insulin resistance induces SREBP-1, leadingto increased fatty acid and triglycerides synthesis. The activation of Srebpsinduces miR-33a and -b expression, leading to decreased HDL cholesterollevels by targeting ABCA1, reduced insulin signaling by targeting IRS2, andreduced cellular β-oxidation by targeting different fatty acid oxidationenzymes. Therapeutic inhibition of miR-33 might result in increased plasmaHDL cholesterol levels, reduced VLDL secretion, and increased insulin signal-ing, thus improving the prognosis of patients with metabolic syndrome.

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lowHDL, and high triglycerides/VLDL. Importantly, we show thatantagonism of endogenous miR-33a and -b up-regulates fatty acidoxidation and response to insulin in hepatocytes, suggesting thatmiR-33a and -b may be an attractive therapeutic target for meta-bolic syndrome. Although our previous work in mice supports thiscontention by showing that inhibition ofmiR-33a and -b effectivelyincreases HDL (14), the functional relevance of the miR-33b/Srepb-1 association cannot easily be determined using traditionalmodels of insulin resistance, such as rats or mice, because theSrebp1 genes of these animal models lack miR-33b.Recently, siRNAs and miRNAs have gained considerable at-

tention as therapeutic targets (29–32). Different strategies havebeen developed to modulate miRNA effects for therapeuticpurposes. Inhibition of miR expression can be achieved by usingantisense oligonucleotides antagomirs or their chemically mod-ified versions, 2’-O-methyl-group (OMe)–modified oligonucleo-tides and locked nucleic acids (LNA) anti-miRs, as well as byinhibiting the production of the mature forms by disrupting theirprocessing (29–32). There is tremendous therapeutic potentialfor the treatment of cardiovascular diseases by either over-expression or inhibition of miRNAs. Our results suggest thatantagonism of endogenous miR-33 may be useful as a thera-peutic strategy for treating metabolic syndrome and nonalcoholicfatty liver disease (NAFLD), which is, by far, the most commonliver ailment. Although the biology of miRNAs regulating lipidmetabolism and insulin signaling is still an exciting frontier incardiovascular medicine, therapeutic miRNA manipulations areemerging as promising players in the treatment of disease. Ad-ditional research and specialty clinical trials, however, areneeded to translate these therapies into clinical practice.

Materials and MethodsA detailed description of procedures is provided in SI Materials and Methods.

Bodipy Staining and Triglyceride Analysis of Fat Body Larvae and Northern BlotAnalysis for miRNAs. Larval starvation was performed as described previously(33). Fat bodies were dissected in PBS, incubated in 1 μg/mL Bodipy 493/503and 10 μg/mL Hoechst 33352 in 1× PBS for 20 min, mounted on a glass slidewith spacers in 50% glycerol in PBS, and imaged by confocal microscopywithin 2 h. Single confocal sections are shown. Triglyceride levels were cal-culated as previously described (32). Small RNA Northern blots were per-formed as described previously (34).

Immunohistochemistry. Huh7 cells were transfected with miR-33 or Con-miRas described above and incubated with 1 mM oleate for 12 h. Then, cellswere washed two times with cold PBS and starved for the next 24 h. Cellswere fixed for 1 h in 4% paraformaldehyde/PBS and stained for 1 h withBodipy 493/503 in PBS. The coverslips were then mounted on glass slideswith Gelvatol/DAPI and analyzed with an epifluorescence microscope(Axiovert; Carl Zeiss MicroImaging) with a 40× objective. Analysis of dif-ferent images was performed using openlab software (Improvision). Inanother set of experiments, we transfected Huh7 cells with miR-33 or Con-miR for 24 h. Then, cells were infected with FoxO1-GFP as previously de-scribed (35) for 24 h and starved for the next 24 h before insulin stimu-lation. We captured microscope images on a fluorescence microscopeusing 488-nm laser excitation for GFP. Images were captured with a 40×objective and analyzed with the Image J software (National Institutes ofHealth). To calculate relative nuclear fluorescence, we divided nuclearfluorescence by the total amount of cellular fluorescence. All quantitativevalues represent averages from at least 30 cells per well. Data are the mean± SEM and are representative of three independent experiments.

ACKNOWLEDGMENTS. We thank Domenico Accili for providing the FoxO1adenovirus construct. This work was supported by the Deutsche Forschungs-gemeinschaft (Exc 257); Neurocure (D.C.-S. and E.E.); American Heart Associ-ation Grants SDG-0835481N (to Y.S.) and SDG-0835585D (to C.F.-H.); NationalInstitutes of Health Grants 1P30HL101270-01, R01HL58541 (to E.A.F.),RO1AG20255 (to K.J.M.), R01GM083300 (to E.C.L.), R01HL16063 andRO1HL107953 (to C.F.-H.); and the Alfred Bressler Scholar Fund (to E.C.L.).

1. Alberti KG, Zimmet P, Shaw J (2006) Metabolic syndrome—a new world-widedefinition. A Consensus Statement from the International Diabetes Federation.Diabet Med 23:469–480.

2. Glass CK, Witztum JL (2001) Atherosclerosis. the road ahead. Cell 104:503–516.3. Hotamisligil GS (2006) Inflammation and metabolic disorders. Nature 444:860–867.4. Lusis AJ (2000) Atherosclerosis. Nature 407:233–241.5. Brown MS, Goldstein JL (1997) The SREBP pathway: Regulation of cholesterol

metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340.

6. Horton JD, Goldstein JL, BrownMS (2002) SREBPs: Activators of the complete programof cholesterol and fatty acid synthesis in the liver. J Clin Invest 109:1125–1131.

7. Osborne TF (2000) Sterol regulatory element-binding proteins (SREBPs): Key regulatorsof nutritional homeostasis and insulin action. J Biol Chem 275:32379–32382.

8. Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355.9. Bartel DP (2009) MicroRNAs: Target recognition and regulatory functions. Cell 136:

215–233.10. Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-

transcriptional regulation by microRNAs: Are the answers in sight? Nat Rev Genet 9:102–114.

11. Gerin I, et al. (2010) Expression of miR-33 from an SREBP2 intron inhibits cholesterolexport and fatty acid oxidation. J Biol Chem 285:33652–33661.

12. Marquart TJ, Allen RM, Ory DS, Baldán A (2010) miR-33 links SREBP-2 induction torepression of sterol transporters. Proc Natl Acad Sci USA 107:12228–12232.

13. Najafi-Shoushtari SH, et al. (2010) MicroRNA-33 and the SREBP host genes cooperateto control cholesterol homeostasis. Science 328:1566–1569.

14. Rayner KJ, et al. (2010) MiR-33 contributes to the regulation of cholesterolhomeostasis. Science 328:1570–1573.

15. Terán-García M, Bouchard C (2007) Genetics of the metabolic syndrome. Appl PhysiolNutr Metab 32:89–114.

16. Rawson RB (2003) The SREBP pathway—insights from Insigs and insects. Nat Rev MolCell Biol 4:631–640.

17. Brady PS, Ramsay RR, Brady LJ (1993) Regulation of the long-chain carnitineacyltransferases. FASEB J 7:1039–1044.

18. Eaton S, et al. (2000) The mitochondrial trifunctional protein: Centre of a beta-oxidation metabolon? Biochem Soc Trans 28:177–182.

19. Mannaerts GP, Van Veldhoven PP, Casteels M (2000) Peroxisomal lipid degradationvia beta- and alpha-oxidation in mammals. Cell Biochem Biophys 32:73–87.

20. Ramsay RR, Gandour RD (1999) Selective modulation of carnitine long-chainacyltransferase activities. Kinetics, inhibitors, and active sites of COT and CPT-II. AdvExp Med Biol 466:103–109.

21. Haeusler RA, Accili D (2008) The double life of Irs. Cell Metab 8:7–9.22. Thirone AC, Huang C, Klip A (2006) Tissue-specific roles of IRS proteins in insulin

signaling and glucose transport. Trends Endocrinol Metab 17:72–78.23. White MF (2002) IRS proteins and the common path to diabetes. Am J Physiol

Endocrinol Metab 283:E413–E422.24. Accili D, Arden KC (2004) FoxOs at the crossroads of cellular metabolism,

differentiation, and transformation. Cell 117:421–426.25. Xiao C, et al. (2010) SIRT6 deficiency results in severe hypoglycemia by enhancing

both basal and insulin-stimulated glucose uptake in mice. J Biol Chem 285:36776–36784.

26. Zhong L, et al. (2010) The histone deacetylase Sirt6 regulates glucose homeostasisvia Hif1alpha. Cell 140:280–293.

27. Kim HS, et al. (2010) Hepatic-specific disruption of SIRT6 in mice results in fatty liverformation due to enhanced glycolysis and triglyceride synthesis. Cell Metab 12:224–236.

28. Brown MS, Ye J, Goldstein JL (2010) Medicine. HDL miR-ed down by SREBP introns.Science 328:1495–1496.

29. Elmén J, et al. (2008) LNA-mediated microRNA silencing in non-human primates.Nature 452:896–899.

30. Elmén J, et al. (2008) Antagonism of microRNA-122 in mice by systemicallyadministered LNA-antimiR leads to up-regulation of a large set of predicted targetmRNAs in the liver. Nucleic Acids Res 36:1153–1162.

31. Esau C, et al. (2006) miR-122 regulation of lipid metabolism revealed by in vivoantisense targeting. Cell Metab 3:87–98.

32. Krützfeldt J, et al. (2005) Silencing of microRNAs in vivo with ‘antagomirs’. Nature438:685–689.

33. Palanker L, Tennessen JM, Lam G, Thummel CS (2009) Drosophila HNF4 regulates lipidmobilization and beta-oxidation. Cell Metab 9:228–239.

34. Okamura K, Hagen JW, Duan H, Tyler DM, Lai EC (2007) The mirtron pathwaygenerates microRNA-class regulatory RNAs in Drosophila. Cell 130:89–100.

35. Frescas D, Valenti L, Accili D (2005) Nuclear trapping of the forkhead transcriptionfactor FoxO1 via Sirt-dependent deacetylation promotes expression of glucogeneticgenes. J Biol Chem 280:20589–20595.

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