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.