miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling - PubMed (original) (raw)

. 2011 May 31;108(22):9232-7.

doi: 10.1073/pnas.1102281108. Epub 2011 May 16.

Leigh Goedeke, Peter Smibert, Cristina M Ramírez, Nikhil P Warrier, Ursula Andreo, Daniel Cirera-Salinas, Katey Rayner, Uthra Suresh, José Carlos Pastor-Pareja, Enric Esplugues, Edward A Fisher, Luiz O F Penalva, Kathryn J Moore, Yajaira Suárez, Eric C Lai, Carlos Fernández-Hernando

Affiliations

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

Alberto Dávalos et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Cellular imbalances of cholesterol and fatty acid metabolism result in pathological processes, including atherosclerosis and metabolic syndrome. Recent work from our group and others has shown that the intronic microRNAs hsa-miR-33a and hsa-miR-33b are located within the sterol regulatory element-binding protein-2 and -1 genes, respectively, and regulate cholesterol homeostasis in concert with their host genes. Here, we show that miR-33a and -b also regulate genes involved in fatty acid metabolism and insulin signaling. miR-33a and -b target key enzymes involved in the regulation of fatty acid oxidation, including carnitine O-octaniltransferase, carnitine palmitoyltransferase 1A, hydroxyacyl-CoA-dehydrogenase, Sirtuin 6 (SIRT6), and AMP kinase subunit-α. Moreover, miR-33a and -b also target the insulin receptor substrate 2, an essential component of the insulin-signaling pathway in the liver. Overexpression of miR-33a and -b reduces both fatty acid oxidation and insulin signaling in hepatic cell lines, whereas inhibition of endogenous miR-33a and -b increases these two metabolic pathways. Together, these data establish that miR-33a and -b regulate pathways controlling three of the risk factors of metabolic syndrome, namely levels of HDL, triglycerides, and insulin signaling, and suggest that inhibitors of miR-33a and -b may be useful in the treatment of this growing health concern.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

Fig. 4.

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

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