miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling

Alberto 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,EricC.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 review February 9, 2011) Cellular imbalances of cholesterol and fatty acid metabolism result stranded regulatory noncoding RNAs are encoded in the ge- in pathological processes, including atherosclerosis and metabolic nome, and most are processed from primary transcripts by the syndrome. Recent work from our group and others has shown sequential actions of Drosha and Dicer enzymes (8–10). In the that the intronic microRNAs hsa-miR-33a and hsa-miR-33b are lo- cytoplasm, mature miRNAs are incorporated into the cytoplas- cated within the sterol regulatory element-binding protein-2 and mic RNA-induced silencing complex (RISC) and bind to par- -1 genes, respectively, and regulate cholesterol homeostasis in tially complementary target sites in the 3′ UTRs of mRNA. concert with their host genes. Here, we show that miR-33a and miRNA targeting of mRNAs inhibits their expression through -b also regulate genes involved in fatty acid metabolism and in- mRNA destabilization, repression of translation, or a combina- sulin signaling. miR-33a and -b target key enzymes involved in tion of both processes (8–10). the regulation of fatty acid oxidation, including carnitine O-octa- We and others provided identification of a highly conserved niltransferase, carnitine palmitoyltransferase 1A, hydroxyacyl-CoA- miRNA family, miR-33, within the intronic sequences of the dehydrogenase, Sirtuin 6 (SIRT6), and AMP kinase subunit-α. More- Srebp genes in organisms ranging from Drosophila to humans over, miR-33a and -b also target the insulin receptor substrate 2, (11–14). Two miR-33 genes are present in humans: miR-33b, an essential component of the insulin-signaling pathway in the which is present in intron 17 of the Srebp-1 gene on chromosome liver. Overexpression of miR-33a and -b reduces both fatty acid 17, and miR-33a, which is located in intron 16 of the Srebp-2 gene oxidation and insulin signaling in hepatic cell lines, whereas in- on chromosome 22. In mice, however, there is only one miR-33 hibition of endogenous miR-33a and -b increases these two met- gene, which is conserved with human miR-33a and located within abolic pathways. Together, these data establish that miR-33a and intron 15 of the mouse Srebp-2 gene. -b regulate pathways controlling three of the risk factors of met- We recently showed that miR-33a is cotranscribed with its host abolic syndrome, namely levels of HDL, triglycerides, and insulin gene Srebp-2 like many intronic miRNAs, and it targets genes in- signaling, and suggest that inhibitors of miR-33a and -b may be volved in cholesterol export, including the adenosine triphosphate useful in the treatment of this growing health concern. binding cassette (ABC) transporters ABCA1 and ABCG1 and the endolysosomal transport protein Niemann-Pick C1 (NPC1) (14). lipid homeostasis | posttranscriptional regulation | cardiovascular disease This regulatory function of miR-33a ensures that the cell is pro- tected under low sterol conditions from additional sterol loss. In any diseases result from perturbations in lipid homeostasis, addition to this role in maintaining cholesterol homeostasis, Mincluding atherosclerosis, type II diabetes, and metabolic we now show that miR-33a and -b also regulate fatty acid metab- syndrome (1–4). The intracellular and membrane levels of fatty olism and insulin signaling. We identify putative binding sites for acids and cholesterol are under constant surveillance and are miR-33 in the 3′ UTR of carnitine O-octaniltransferase (CROT), coordinated with de novo lipid biosynthesis by endoplasmic re- carnitine palmitoyltransferase 1A (CPT1a), hydroxyacyl-CoA-de- ticulum (ER)-bound sterol regulatory element-binding proteins hydrogenase (HADHB), AMP kinase subunit-α (AMPKα), and (SREBPs) (5–7). The SREBP family of basic helix–loop–helix– insulin receptor substrate 2 (IRS2) and show that miR-33a and -b leucine zipper (bHLH-LZ) transcription factors consists of specifically inhibit the expression of these genes. The physiological SREBP-1a, SREBP-1c, and SREBP-2 proteins that are encoded relevance of this targeting is revealed by miR-33 overexpression in by two unique genes, Srebp-1 and Srebp-2 (5–7). The SREBPs hepatic cells, which reduces both fatty acid oxidation and insulin differ in their tissue-specific expression, their target gene selec- signaling. Furthermore, inhibition of endogenous miR-33 increa- α tivity, and the relative potencies of their trans-activation domains. ses the expression of CROT, CPT1a, HADHB, AMPK , and IRS2 SREBP-1c regulates the transcription of genes involved in fatty and up-regulates fatty acid oxidation and insulin signaling. To- acid 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), 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. contributed which catalyzes a rate-limiting step in cholesterol biosynthesis, new 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., and the low-density lipoprotein receptor (LDLr), which imports 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. – cholesterol from the blood (5 7). Increased SREBP activity The authors declare no conflict of interest. causes cholesterol and fatty acid accumulation and down-regu- This article is a PNAS Direct Submission. J.L.W. is a guest editor invited by the Editorial lates the SCAP/SREBP pathway by feedback inhibition. In this Board. way, lipid metabolism within cells is tightly regulated. 1A.D. and L.G. contributed equally to this work. In addition to classical transcriptional regulators, a class of 2To whom correspondence should be addressed. E-mail: carlos.fernandez-hernando@ noncoding RNAs, termed microRNAs (miRNAs), has emerged nyumc.org. as critical regulators of gene expression acting predominantly at This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the posttranscriptional level (8–10). These short (22 nt) double- 1073/pnas.1102281108/-/DCSupplemental.

9232–9237 | PNAS | May 31, 2011 | vol. 108 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1102281108 Downloaded by guest on October 1, 2021 gether, these data suggest that feedback loops involving SREBPs of the mammalian peroxisome to the cytosol and mitochondria and miR-33a and -b balance cholesterol metabolism, fatty acid (17–20). CPT1a is a mitochondrial enzyme that mediates the oxidation, and insulin signaling, three of the major risk factors of transport of long fatty acids across the membrane by binding them metabolic syndrome (1, 3, 15). to carnitine, and it is the rate-limiting enzyme that regulates fatty acid oxidation (17–20). HADHB is the β-subunit of the mito- Results chondrial trifunctional protein, which catalyzes the last three steps miR-33 Targets Genes Regulating β-Oxidation of Fatty Acid and of mitochondrial β-oxidation of long-chain fatty acids, whereas Insulin Signaling. We have previously described the presence of AMPKα stimulates hepatic fatty acid oxidation and ketogenesis miR-33a in the Srebp-2 gene. miR-33a is found within the same (17–20). Interestingly, we also identified IRS2, a component of the intron of Srebp-2 from many animal species, including large and insulin-signaling pathway, as a potential target of miR-33. small mammals, chickens, and frogs. Interestingly, the fruit fly D. To determine whether miR-33b targets these predicted target melanogaster also has a highly conserved mature form of miR-33a, genes, we generated reporter constructs with the luciferase but these organisms do not synthesize sterols. SREBP in flies coding sequence fused to the 3′ UTRs of these genes. miR-33b regulates fatty acid metabolism (16), which is reminiscent of the markedly repressed the activity of the Crot, Cpt1a, Hadhb, function of the Srebp-1 gene in mammals (6). As shown in Fig. S1 Ampkα, and Irs2 3′ UTR luciferase constructs (Fig. S3). Fur- A and B, miR-33b is synchronously expressed with SREBP-1c in thermore, mutation of the miR-33 target sites in these constructs human hepatic Huh7 cells treated with an agonist of the liver X relieved miR-33b repression of the 3′ UTR of Crot, Cpt1a, receptor (LXR), a transcriptional regulator of Srebp-1c expres- Hadhb, Ampkα, and Irs2, consistent with a direct interaction of sion. Kinetic analysis of miR-33b induction revealed a concomi- miR-33b with these sites (Fig. S3). Mutation of both miR-33 sites tant increase in miR-33b and SREBP-1c expression, consistent in the 3′ UTR of Crot was necessary to completely reverse the with their coregulation. Thus, we postulated that miR-33a and -b, inhibitory effects of miR-33 (Fig. S3). which differ only in 2 nt (Fig. S1C), might, therefore, regulate We next determined the effect of miR-33 on mRNA and protein genes involved in lipid metabolism. To search for potential tar- expression of CROT, CPT1a, HADHB, AMPKα,IRS2,andlipid- gets, we performed gene ontology and biological association related genes that lack predicted miR-33 binding sites. Transfection analyses using Pathway Studio 7 software (Ariadne Genomics) of Huh7 cells with miR-33b (32-fold increase expression) signifi- and looked for enrichment of specific target genes associated with cantly inhibited the mRNA levels of CROT, CPT1a, HADHB, lipid metabolism. As shown in Fig. S2 (red box), several genes AMPKα,andIRS2 (Fig. 1A). Notably, inhibition of endogenous involved in fatty acid β-oxidation have predicted targets for miR- miR-33b using anti–miR-33b oligonucleotides (threefold decrease 33, including CROT, CPT1a, HADHB and AMPKα (17–20). expression) increased the mRNA expression of CROT, CPT1a, CROT, a carnitine acyltransferase that catalyzes the reversible AMPKα,andIRS2 in Huh7 cells (Fig. 1B), consistent with a physi- transfer of fatty acyl between CoA and carnitine, provides a cru- ological role for miR-33b in regulating the expression of these cial step in the transport of medium and long-chain acyl-CoA out genes. Similar regulation of these genes by miR-33b was also seen

A B E miR-33b inh-33b 1.5 2.5 * 1.2 2.0 *

0.9 1.5 * * * * 0.6 * * 1.0 0.3 0.5 mRNA (fold change) mRNA MEDICAL SCIENCES mRNA (fold change) mRNA 0 0

C D 1.5 Con-inh Con-miR miR-33b 2.0 Con miR Con-inh inh-33b inh-33b miR-33b * IRS2 IRS2 * 1.5 1.0 * * * AMPK1 AMPK1 CROT 1.0 * CROT CPT1a * CPT1a 0.5 * * HADHB 0.5 * HADHB

HSP90 Relative expression (a.u) HSP90 0 0

Fig. 1. Posttranscriptional regulation of IRS2, AMPKα, CROT, CPT1a, and HADHB by miR-33b. Quantitative RT-PCR expression profile of selected miR-33 predicted target and other related genes in human hepatic Huh7 cell line (A) after overexpressing miR-33b and (B) after endogenous inhibition of miR-33b by anti–miR-33b. Western blot analysis of HepG2 cells (C) overexpressing or (D) inhibiting endogenous miR-33b. Heat shock protein (HSP)90 bands are the loading control. (E) Specificity of miR-33b on fatty acid metabolism-related genes. qRT-PCR array analysis of fatty acid metabolism-related genes from HepG2 cells 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.

Dávalos et al. PNAS | May 31, 2011 | vol. 108 | no. 22 | 9233 Downloaded by guest on October 1, 2021 at the protein level (Fig. 1 C and D). We next investigated the effect but differ in 2 of 19 nt of the mature RNA (Fig. S1C). To de- of manipulating miR-33 levels in vivo in mice using lentiviruses termine whether miR-33a and -b have similar effects on CROT, encoding premiR-33, anti–miR-33, or a control. Efficient lentiviral CPT1a, HADHB, AMPKα, Sirtuin 6 (SIRT6), and IRS2 protein delivery was previously confirmed (14). Consistent with our in vitro expression, we transfected Huh7 cells with a control miR, miR- results, miR-33 reduced hepatic CROT, HADHB, CPT1a, IRS2, 33a, or -b. As seen in Fig. S6, miR-33a and -b inhibited CROT, and ABCA1 mRNA expression (Fig. S4). Conversely, mice CPT1a, HADHB, AMPKα, SIRT6, and IRS2 protein expression expressing anti–miR-33 showed a modest increase of CROT, to a similar extent. In addition, both miR-33a and -b significantly CPT1a, IRS2,andABCA1 mRNA expression, although the effect inhibited the 3′ UTR activity of Crot, Cpt1a, Hadhb, Ampkα, and was not statistically significant (Fig. S4). Irs2 with only modest differences, indicating that the 2-nt vari- To show the specificity of miR-33b targeting of CROT, CPT1a, ation in the mature forms of miR-33a and -b does not appre- HADHB, AMPKα,andIRS2, we assessed the effect of miR-33b ciably affect the gene targeting by these miRNAs. overexpression in HepG2 cells on other fatty acid metabolism- related genes using an array that included 84 genes involved in miR-33 Inhibits Cellular Fatty Acid Oxidation. To evaluate the effects fatty acid transport and biosynthesis, ketogenesis, and ketone body of miR-33a and -b on fatty acid β-oxidation, we measured the 14 14 metabolism. Whereas CROT, CPT1a,andAMPKα were pre- release of [ C]-carbon dioxide from the oxidation of [ C]-oleate. dictably down-regulated by miR-33, we did not observe changes in miR-33b overexpression (27-fold increase) markedly reduced the the expression of non–miR-33 targets (Fig. 1E). Furthermore, fatty acid β-oxidation in Huh7 (Fig. 2A) cells. Conversely, in- other genes containing putative miR-33 binding sites such as cit- hibition of endogenous miR-33b expression using anti–miR-33 rate synthase (CS)andHMGCR were not affected either at the (2.6-fold increase) increased the rate of fatty acid β-oxidation mRNA level (Fig. S5A)orby3′ UTR activity (Fig. S5B). (Fig. 2B). We next evaluated the accumulation of neutral lipids and lipid droplet formation in Huh7 cells incubated with oleate miR-33a and miR-33b Have Similar Targeting Effects on Genes for 24 h and then starved for the next 24 h. In agreement with the Regulating Cholesterol Metabolism, β-Oxidation of Fatty Acids, and reduced fatty acid β-oxidation rates observed in hepatic cells Insulin Signaling. miR-33a and -b have identical seed sequences overexpressing miR-33b, Huh7 cells transfected with miR-33b

A BC con-miR miR-33b

D E F 60

25 * - 40 miR-33 0h 80 Ins 30 20 24h 20 15 60 30 10 40 miR-8 20

µ g TG/mg prot 5 20 30 2S rRNA

Cell TG (cpm/mg prot) Cell 0 Control miR-33b Control miR-33b

G H 0h 1.2 * 24h 1 0.8 0.6 0.4

mg TG/mg prot 0.2 0 Cg-DsRed Cg-DsRed miR-33 Cg-DsRed Cg-DsRed miR-33

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 transfected with 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 triglyceride synthesis 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, and 2S 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 of transgenic Drosophila overexpressing miR-33 or control transgene in the fat body before and after starvation.

9234 | www.pnas.org/cgi/doi/10.1073/pnas.1102281108 Dávalos et al. Downloaded by guest on October 1, 2021 accumulated more triglycerides (TAG) in larger lipid droplets signaling, we analyzed the effect of miR-33 overexpression on (Fig. 2 C and D). The increase in triglyceride content was in- two of the main downstream effectors of IRS2: the PI3K/AKT dependent of changes in triglyceride synthesis rates. As seen in and rat sarcoma (RAS)/RAF/ERK pathways (21–23). As seen in Fig. 2E, miR-33 expression did not alter basal and insulin-in- Fig. 3 A and B, Huh7 cells transfected with miR-33b showed duced triglyceride synthesis. reduced AKT and ERK phosphorylation after insulin stimulation, Because CPT1a is also a target of miR-33 in Drosophila,weset indicative of reduced IRS2 function. Similar results were ob- out to determine if miR-33 plays a role in maintaining lipid ho- served when we analyzed the AKT activation using an in vitro meostasis in Drosophila. To do this, we generated a transgenic fly kinase assay (Fig. 3C). To determine whether or not IRS2 over- that overexpressed miR-33 in the fat body (Fig. 2F). We hypoth- expression rescues the miR-33 overexpression effect on AKT esized that miR-33–overexpressing flies would retain TAG and phosphorylation, we transfected Huh7 cells with IRS2 cDNA that fatty acids upon starvation as a consequence of reduced fatty acid lacked the 3′ UTR sequence. As seen in Fig. 3 D and E,IRS2 oxidation, which should manifest as an increase in lipid storage. expression rescued the AKT activation on insulin stimulation in Accordingly, fat bodies from starved miR-33 transgenic flies, dis- miR-33b–overexpressing cells. sected and stained with Bodipy, showed large lipid droplets in To gain insights into the role of miR-33 in regulating insulin many cells compared with control flies (Fig. 2G). Similarly, flies signaling, we assessed the effect of miR-33b on 2-deoxyglucose overexpressing miR-33 accumulated more TAG upon starvation uptake after insulin stimulation. As seen in Fig. 3F, miR-33b (Fig. 2H), indicating that the function of miR-33 in regulating fatty overexpression reduced insulin-induced 2-deoxyglucose (2-DOG) acid oxidation is conserved from Drosophila to humans. uptake in Huh7 cells. We also assessed FoxO1 cellular local- ization in insulin-stimulated cells. FoxO1 is a well-defined target miR-33 Regulates Insulin Signaling. To further explore our obser- downstream of the conserved insulin/target of rapamycin (TOR) vation that miR-33 inhibits IRS2 expression, we next assessed the signaling network that has important roles in the regulation of effect of miR-33 on insulin signaling. IRS2 is a cytoplasmic sig- processes as diverse as cellular growth, stress resistance, and en- naling molecule that mediates the effects of insulin, insulin-like ergy homeostasis (24). To date, several proteins are known to growth factor 1, and other cytokines by acting as a molecular interact with FoxO transcription factors, regulating their in- adaptor between receptor tyrosine kinases and downstream tracellular localization and/or activity. One of the best docu- effectors (21–23). To test the role of miR-33 in regulating insulin mented is the AKT/protein kinase B (PKB) kinase, which

A Con-miR miR-33b B C Con-miR miR-33b ABCA1 p-GSK3 / (Ser-219) IR- Total total-AKT 1.2 IRS1 * Lysate HSP90 IRS2 Insulin - + + - + + * Time (min) - 5 15 - 5 15 p-AKTSer473 0.8 Con-miR miR-33b NT total-AKT (a.u) /total-AKT D p-ERK 0.4 IRS2 Ser473 ERK p-AKTSer473

HSP90 p-AKT 0 total-AKT Insulin - + + - + + Insulin - + + - + + MEDICAL SCIENCES Time (min) - 5 15 - 5 15 Time (min) - 5 15 - 5 15 HSP90 Con-miR miR-33b Insulin - + + - + + - Time (min) - 5 15 - 5 15 -

- E 1.5 FG Ins 0.3 ACACA * RRAS2 1 300

-DeltaCt) PPARG 0.03 SORBS1 FRS2 /total-AKT (a.u) (a.u) /total-AKT DOK3 200 AKT3

Ser473 0.5 0.003 FBP1 UCP1 IRS2 100 p-AKT 0.0003 0 PRL

Insulin - + + - + + - Log 10 (miR-33b 2

2-DOG uptake (% of control) 2-DOG uptake (% of control) 0 GCK Time (min) - 5 15 - 5 15 - Con-miR miR-33b 3E-05 Con-miR miR-33b 3E-05 0.0003 0.003 0.03 0.3 IRS2 Log 10 (Control 2 -DeltaCt)

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 AKT kinase assay of postinsulin-stimulated and immunoprecipitated total AKT from Huh7 cells. GSK-3 fusion protein was 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 overexpression reduces 2-deoxyglucose (2-DOG) uptake in Huh7 cells treated with insulin. (G) Quantitative RT-PCR array analysis of insulin signaling related genes from Huh7 cells 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.

Dávalos et al. PNAS | May 31, 2011 | vol. 108 | no. 22 | 9235 Downloaded by guest on October 1, 2021 phosphorylates FoxO in three conserved sites, leading to FoxO metabolism genes. Notably, we show that miR-33a and miR-33b cytoplasmatic retention and transcriptional inactivation. To test have overlapping gene targets, suggesting that cotranscription of the effect of miR-33 on FoxO1 localization, we transfected Huh7 Srebp-2 and miR-33a or Srebp-1 and miR-33b would be predicted with a con-miR or miR-33b and transduced the cells with to regulate both cholesterol metabolism and fatty acid oxidation; a FoxO1-GFP adenovirus. FoxO1-GFP localized primarily to the this establishes a model of reciprocal regulation of cholesterol nucleus in starved cells transfected with Con-miR (Fig. S7A Up- and fatty acid metabolism by SREBPs (Fig. 4). per) or miR-33b (Fig. S7B Upper). Treatment with insulin induced The presence of miR-33a in the intron of Srebp-2 is remarkably FoxO1-GFP translocation from the nucleus to the cytoplasm in conserved in many species, including the fruit fly D. melanogaster. Con-miR–transfected cells (Fig. S7A Lower), whereas cells This was notable, because Drosophila neither synthesizes sterols transfected with miR-33b had the cellular distribution reversed nor expresses ABCA1. Interestingly, the Srebp gene of Drosophila (Fig. S7B Lower). Interestingly, IRS2 overexpression rescued the controls fatty acid production, and our bioinformatic analysis of effect of miR-33 overexpression on FoxO1 intracellular localiza- miR-33a and -b target genes revealed an enrichment of genes tion (Fig. S7 C and D). Together, these experiments identify miR- involved in fatty acid metabolism. We identify herein six miR-33 33 as an important regulator of the insulin-signaling pathways. target genes that regulate fatty acid metabolism and insulin sig- In addition, we also assessed the effect of miR-33b over- naling, including CPT1a, CROT, HADHB, AMPKα, SIRT6, and expression in Huh7 cells on other insulin-related genes using an IRS2. Importantly, we show that miR-33 overexpression reduces array that included 84 genes involved in carbohydrate and lipid fatty acid oxidation in hepatic cell lines. While this manuscript metabolism and target genes for insulin signaling. As expected, was in preparation, Gerin et al. (11) reported similar effects of IRS2 was down-regulated in Huh7 cells transfected with miR-33b miR-33 on CROT, CPT1a, and HADHB expression. Finally, we (Fig. 3D). Moreover, glucokinase (GCK), fibroblast growth factor show that miR-33 transgenic flies show increased lipid accumu- receptor substrate 2 (FRS2), acetyl-CoA carboxylase-α (ACACA), lation in tissues, suggesting that miR-33 regulation of fatty acid and peroxisome proliferator-activated–γ (PPARG) were also metabolism pathways is evolutionarily conserved. inhibited (Fig. 3D). Interestingly, FRS2 is also a predicted target Our work also identifies miR-33a and -b as regulators of insulin of miR-33a and -b. FRS2 has been suggested to participate in signaling. By inhibiting expression of IRS2 in hepatic cells, miR- insulin signaling by recruiting Src-homology-phosphatase 2 (SHP2) 33a and -b reduce the activation of downstream insulin signaling and hence, could function as a docking molecule similar to insulin pathways, including AKT and ERK. Although our previous work receptor substrate proteins. established that miR-33a and Srebp-2 are cotranscribed during In addition to IRS2 and FRS2, our bioinformatic analysis iden- states of cholesterol depletion (14), the changes in Srebp-2 and tified a third miR-33 predicted target involved in glucose homeo- miR-33a transcription were quite small in this setting. By contrast, stasis: the histone deacetylase SIRT6 (25, 26). SIRT6-deficient Srebp-1c is transcribed at extremely high levels in response to in- mice develop normally but succumb to lethal hypoglycemia early in sulin (6, 28), which may be particularly relevant in the setting of life, suggesting an important role of SIRT6 in regulating glucose metabolic syndrome, where insulin resistance is accompanied by metabolism (25, 26). Interestingly, it has also been recently repor- increased triglycerides and plasma levels of very low density lipo- ted that hepatic-specific disruption of SIRT6 in mice results in fatty protein (VLDL) as well as reduced HDL levels (1, 3). Our data liver formation because of enhanced glycolysis and triglyceride indicate that miR-33a and -b impact pathways influencing three of synthesis (27). To confirm that miR-33b targets SIRT6, we cloned the primary risk factors in this disease, namely insulin resistance, the Sirt6 3′ UTR into a luciferase reporter construct. miR-33b markedly repressed the 3′ UTR activity of Sirt6 (Fig. S8A), and mutation of the miR-33 target sites in the 3′ UTR relieved miR-33b repression of Sirt6, consistent with a direct interaction of miR-33b with these sites (Fig. S8A). Furthermore, miR-33b overexpression significantly inhibited the SIRT6 mRNA and protein levels in Huh7 cells (Fig. S8 B and C), whereas inhibition of endogenous miR-33b by anti–miR-33 increased the expression of SIRT6 (Fig. S8 B and C). Although these data are consistent with a physiological role for miR-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 modestly decreased fatty acid β-oxidation (Fig. S8D). Additional experiments, including RIPseq and proteomics, are warranted to understand the miR-33 regulatory network and its implication in lipid and carbohydrate metabolism. Discussion We and others have recently established that, during sterol- limited states, miR-33a is coincidentally generated with Srebp-2 transcription and works to increase cellular cholesterol levels by limiting cholesterol export through the down-regulation of ABC transporters, ABCA1 and ABCG1 (12–14). Importantly, these Fig. 4. Potential role of SREBPs and miR-33a and -b in metabolic syndrome. pathways regulate circulating HDL levels through their roles in In hepatocytes, conditions of low intracellular cholesterol (or statins) induce HDL biogenesis and cellular cholesterol efflux (12–14). We now SREBP-2, leading to increased lipoprotein uptake and endogenous cholesterol show that a second member of the miR-33 family, miR-33b, is biosynthesis. Hyperinsulinemia or insulin resistance induces SREBP-1, leading Srebps coregulated with the human Srebp1 gene and targets genes in- to increased fatty acid and triglycerides synthesis. The activation of induces miR-33a and -b expression, leading to decreased HDL cholesterol volved in fatty acid oxidation and insulin signaling. Together, levels by targeting ABCA1, reduced insulin signaling by targeting IRS2, and these results suggest a paradigm in which miR-33a and miR-33b reduced cellular β-oxidation by targeting different fatty acid oxidation act in concert with their host genes, Srebp-2 and Srebp-1, to boost enzymes. Therapeutic inhibition of miR-33 might result in increased plasma intracellular cholesterol and fatty acid levels by balancing tran- HDL cholesterol levels, reduced VLDL secretion, and increased insulin signal- scriptional induction and posttranscriptional repression of lipid ing, thus improving the prognosis of patients with metabolic syndrome.

9236 | www.pnas.org/cgi/doi/10.1073/pnas.1102281108 Dávalos et al. Downloaded by guest on October 1, 2021 low HDL, and high triglycerides/VLDL. Importantly, we show that Bodipy Staining and Triglyceride Analysis of Fat Body Larvae and Northern Blot antagonism of endogenous miR-33a and -b up-regulates fatty acid Analysis for miRNAs. Larval starvation was performed as described previously oxidation and response to insulin in hepatocytes, suggesting that (33). Fat bodies were dissected in PBS, incubated in 1 μg/mL Bodipy 493/503 miR-33a and -b may be an attractive therapeutic target for meta- and 10 μg/mL Hoechst 33352 in 1× PBS for 20 min, mounted on a glass slide bolic syndrome. Although our previous work in mice supports this with spacers in 50% glycerol in PBS, and imaged by confocal microscopy contention by showing that inhibition of miR-33a and -b effectively within 2 h. Single confocal sections are shown. Triglyceride levels were cal- increases HDL (14), the functional relevance of the miR-33b/ culated as previously described (32). Small RNA Northern blots were per- Srepb-1 association cannot easily be determined using traditional formed as described previously (34). models of insulin resistance, such as rats or mice, because the Srebp1 genes of these animal models lack miR-33b. Immunohistochemistry. Huh7 cells were transfected with miR-33 or Con-miR Recently, siRNAs and miRNAs have gained considerable at- as described above and incubated with 1 mM oleate for 12 h. Then, cells – were washed two times with cold PBS and starved for the next 24 h. Cells tention as therapeutic targets (29 32). Different strategies have fi been developed to modulate miRNA effects for therapeutic were xed for 1 h in 4% paraformaldehyde/PBS and stained for 1 h with purposes. Inhibition of miR expression can be achieved by using Bodipy 493/503 in PBS. The coverslips were then mounted on glass slides with Gelvatol/DAPI and analyzed with an epifluorescence microscope antisense oligonucleotides antagomirs or their chemically mod- (Axiovert; Carl Zeiss MicroImaging) with a 40× objective. Analysis of dif- ified versions, 2’-O-methyl-group (OMe)–modified oligonucleo- ferent images was performed using openlab software (Improvision). In tides and locked nucleic acids (LNA) anti-miRs, as well as by another set of experiments, we transfected Huh7 cells with miR-33 or Con- inhibiting the production of the mature forms by disrupting their – miR for 24 h. Then, cells were infected with FoxO1-GFP as previously de- processing (29 32). There is tremendous therapeutic potential scribed (35) for 24 h and starved for the next 24 h before insulin stimu- for the treatment of cardiovascular diseases by either over- lation. We captured microscope images on a fluorescence microscope expression or inhibition of miRNAs. Our results suggest that using 488-nm laser excitation for GFP. Images were captured with a 40× antagonism of endogenous miR-33 may be useful as a thera- objective and analyzed with the Image J software (National Institutes of peutic strategy for treating metabolic syndrome and nonalcoholic Health). To calculate relative nuclear fluorescence, we divided nuclear fatty liver disease (NAFLD), which is, by far, the most common fluorescence by the total amount of cellular fluorescence. All quantitative liver ailment. Although the biology of miRNAs regulating lipid values represent averages from at least 30 cells per well. Data are the mean metabolism and insulin signaling is still an exciting frontier in ± SEM and are representative of three independent experiments. cardiovascular medicine, therapeutic miRNA manipulations are emerging as promising players in the treatment of disease. Ad- ACKNOWLEDGMENTS. We thank Domenico Accili for providing the FoxO1 ditional research and specialty clinical trials, however, are adenovirus construct. This work was supported by the Deutsche Forschungs- needed to translate these therapies into clinical practice. 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.); National Institutes of Health Grants 1P30HL101270-01, R01HL58541 (to E.A.F.), Materials and Methods RO1AG20255 (to K.J.M.), R01GM083300 (to E.C.L.), R01HL16063 and A detailed description of procedures is provided in SI Materials and Methods. RO1HL107953 (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-wide 20. Ramsay RR, Gandour RD (1999) Selective modulation of carnitine long-chain definition. A Consensus Statement from the International Diabetes Federation. acyltransferase activities. Kinetics, inhibitors, and active sites of COT and CPT-II. Adv Diabet Med 23:469–480. Exp Med Biol 466:103–109. 2. Glass CK, Witztum JL (2001) Atherosclerosis. the road ahead. Cell 104:503–516. 21. Haeusler RA, Accili D (2008) The double life of Irs. Cell Metab 8:7–9. 3. Hotamisligil GS (2006) Inflammation and metabolic disorders. Nature 444:860–867. 22. Thirone AC, Huang C, Klip A (2006) Tissue-specific roles of IRS proteins in insulin 4. Lusis AJ (2000) Atherosclerosis. Nature 407:233–241. signaling and glucose transport. Trends Endocrinol Metab 17:72–78. 5. Brown MS, Goldstein JL (1997) The SREBP pathway: Regulation of cholesterol 23. White MF (2002) IRS proteins and the common path to diabetes. Am J Physiol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89: Endocrinol Metab 283:E413–E422. 331–340. 24. Accili D, Arden KC (2004) FoxOs at the crossroads of cellular metabolism, 6. Horton JD, Goldstein JL, Brown MS (2002) SREBPs: Activators of the complete program differentiation, and transformation. Cell 117:421–426. J Clin Invest – of cholesterol and fatty acid synthesis in the liver. 109:1125 1131. 25. Xiao C, et al. (2010) SIRT6 deficiency results in severe hypoglycemia by enhancing

7. Osborne TF (2000) Sterol regulatory element-binding proteins (SREBPs): Key regulators both basal and insulin-stimulated glucose uptake in mice. J Biol Chem 285: MEDICAL SCIENCES JBiolChem – of nutritional homeostasis and insulin action. 275:32379 32382. 36776–36784. Nature – 8. Ambros V (2004) The functions of animal microRNAs. 431:350 355. 26. Zhong L, et al. (2010) The histone deacetylase Sirt6 regulates glucose homeostasis 9. Bartel DP (2009) MicroRNAs: Target recognition and regulatory functions. Cell 136: via Hif1alpha. Cell 140:280–293. 215–233. 27. Kim HS, et al. (2010) Hepatic-specific disruption of SIRT6 in mice results in fatty liver 10. Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post- formation due to enhanced glycolysis and triglyceride synthesis. Cell Metab 12: transcriptional regulation by microRNAs: Are the answers in sight? Nat Rev Genet 9: 224–236. 102–114. 28. Brown MS, Ye J, Goldstein JL (2010) Medicine. HDL miR-ed down by SREBP introns. 11. Gerin I, et al. (2010) Expression of miR-33 from an SREBP2 intron inhibits cholesterol Science 328:1495–1496. export and fatty acid oxidation. J Biol Chem 285:33652–33661. 29. Elmén J, et al. (2008) LNA-mediated microRNA silencing in non-human primates. 12. Marquart TJ, Allen RM, Ory DS, Baldán A (2010) miR-33 links SREBP-2 induction to Nature 452:896–899. repression of sterol transporters. Proc Natl Acad Sci USA 107:12228–12232. 30. Elmén J, et al. (2008) Antagonism of microRNA-122 in mice by systemically 13. Najafi-Shoushtari SH, et al. (2010) MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis. Science 328:1566–1569. administered LNA-antimiR leads to up-regulation of a large set of predicted target Nucleic Acids Res – 14. Rayner KJ, et al. (2010) MiR-33 contributes to the regulation of cholesterol mRNAs in the liver. 36:1153 1162. homeostasis. Science 328:1570–1573. 31. Esau C, et al. (2006) miR-122 regulation of lipid metabolism revealed by in vivo Cell Metab – 15. Terán-García M, Bouchard C (2007) Genetics of the metabolic syndrome. Appl Physiol antisense targeting. 3:87 98. ‘ ’ Nature Nutr Metab 32:89–114. 32. Krützfeldt J, et al. (2005) Silencing of microRNAs in vivo with antagomirs . 16. Rawson RB (2003) The SREBP pathway—insights from Insigs and insects. Nat Rev Mol 438:685–689. Cell Biol 4:631–640. 33. Palanker L, Tennessen JM, Lam G, Thummel CS (2009) Drosophila HNF4 regulates lipid 17. Brady PS, Ramsay RR, Brady LJ (1993) Regulation of the long-chain carnitine mobilization and beta-oxidation. Cell Metab 9:228–239. acyltransferases. FASEB J 7:1039–1044. 34. Okamura K, Hagen JW, Duan H, Tyler DM, Lai EC (2007) The mirtron pathway 18. Eaton S, et al. (2000) The mitochondrial trifunctional protein: Centre of a beta- generates microRNA-class regulatory RNAs in Drosophila. Cell 130:89–100. oxidation metabolon? Biochem Soc Trans 28:177–182. 35. Frescas D, Valenti L, Accili D (2005) Nuclear trapping of the forkhead transcription 19. Mannaerts GP, Van Veldhoven PP, Casteels M (2000) Peroxisomal lipid degradation factor FoxO1 via Sirt-dependent deacetylation promotes expression of glucogenetic via beta- and alpha-oxidation in mammals. Cell Biochem Biophys 32:73–87. genes. J Biol Chem 280:20589–20595.

Dávalos et al. PNAS | May 31, 2011 | vol. 108 | no. 22 | 9237 Downloaded by guest on October 1, 2021