RESUMEN
BACKGROUND: Hepatic expression of microRNA-107 is ubiquitously upregulated in various metabolic diseases. In our previous study, we had demonstrated that fatty acid synthase (FASN) is a target of miR-107. miR-107-FASN interaction, by inducing endoplasmic reticulum (ER) stress, promotes lipid accumulation in hepatocytes. Here, we explore the possible mechanism(s) of the miR-107-FASN-ER stress on hepatic lipid metabolism. METHODS: HepG2 cells were transfected with the scramble or miR-107 and/or its inhibitor. Transcript levels of lipid droplet membrane proteins, apolipoproteins and ß-oxidation genes were quantified by quantitative reverse transcription-PCR. Cells were treated with tunicamycin (Tm, 1 h) and 4-PBA (4-phenyl butyric acid, 8 h) or transfected with hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, apha subunit (HADHA) short interfering RNA or its overexpression vector. Mice were injected with the scramble or mmu-miR-107 (2.5 mg kg(-1) body weight) and random glucose levels were measured and oral glucose tolerance test was performed. Serum levels of cholesterol, triglyceride and serum glutamic oxaloacetic transaminase/serum glutamic-pyruvic transaminase (SGOT/SGPT) were evaluated. Hepatic tissues were collected to estimate levels of miR-107 and mitochondrial ß-oxidation genes. Six-micrometer-thick cryosections of hepatic tissues were prepared and stained with Oil Red O for lipid accumulation. RESULTS: miR-107 does not alter the expression of lipid metabolism-related transcription factors, lipid droplet components and apolipiprotein B. miR-107 significantly decreased the levels of the mitochondrial ß-oxidation enzyme, HADHA in HepG2 cells (P<0.01), which was prevented by the miR-107 inhibitor. Similar decrease was observed with Tm (P<0.001), suggesting that HADHA inhibition is promoted by ER stress induction. Interestingly, miR-107-mediated HADHA suppression was rescued by the ER stress inhibitor, 4-PBA (P<0.01). HADHA overexpression rescued miR-107-induced lipid accumulation (P<0.01). miR-107 injection in mice increased random blood glucose levels (P<0.05) and impaired glucose tolerance (P<0.05). Hepatic levels of Hadha were significantly decreased (P<0.001 and P<0.05) accompanied by increased lipid accumulation (P<0.001). CONCLUSIONS: miR-107 promotes hepatic lipid accumulation by suppressing transcript levels of HADHA, induces hyperglycemia and impairs glucose tolerance. We conclude that miR-107 regulation of fatty acid oxidation is an important contributor toward hepatic lipid accumulation.
Asunto(s)
Estrés del Retículo Endoplásmico , Intolerancia a la Glucosa , Metabolismo de los Lípidos , Hígado/metabolismo , MicroARNs , Mitocondrias/genética , Mitocondrias/metabolismo , Animales , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Regulación de la Expresión Génica , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Hepatocitos/metabolismo , Humanos , Inmunohistoquímica , Lipogénesis/fisiología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Oxidación-ReducciónRESUMEN
The fluorescence studies of E. coli porin OmpF at pH 7.5 were carried out using excitation at 280 and 305 nm. Similar studies were performed in presence of a denaturant (urea) and a quencher (KI). Results show that both the tryptophans present in OmpF (residue numbers 61 and 214) contribute to fluorescence at 280 nm excitation, whereas only one residue shows fluorescence emission when excited at 305 nm. Based on these findings and the available crystal structure, it is speculated that tryptophan 61 of OmpF is selectively excited at 305 nm. The present studies point out some interesting features of the tryptophan microenvironments in OmpF.
