RESUMO
The anti-obesity effect of conjugated linoleic acid (CLA) has been well elucidated, but whether CLA affects fat deposition by regulating intestinal dietary fat absorption remains largely unknown. Thus, this study aimed to investigate the effects of CLA on intestinal fatty acid uptake and chylomicron formation and explore the possible underlying mechanisms. We found that CLA supplementation reduced the intestinal fat absorption in HFD (high fat diet)-fed mice accompanied by the decreased serum TG level, increased fecal lipids and decreased intestinal expression of ApoB48 and MTTP. Correspondingly, c9, t11-CLA, but not t10, c12-CLA induced the reduction of fatty acid uptake and TG content in PA (palmitic acid)-treated MODE-K cells. In the mechanism of fatty acid uptake, c9, t11-CLA inhibited the binding of CD36 with palmitoyltransferase DHHC7, thus leading to the decreases of CD36 palmitoylation level and localization on the cell membrane of the PA-treated MODE-K cells. In the mechanism of chylomicron formation, c9, t11-CLA inhibited the formation of the CD36/FYN/LYN complex and the activation of the ERK pathway in the PA-treated MODE-K cells. In in vivo verification, CLA supplementation reduced the DHHC7-mediated total and cell membrane CD36 palmitoylation and suppressed the formation of the CD36/FYN/LYN complex and the activation of the ERK pathway in the jejunum of HFD-fed mice. Altogether, these data showed that CLA reduced intestinal fatty acid uptake and chylomicron formation in HFD-fed mice associated with the inhibition of DHHC7-mediated CD36 palmitoylation and the downstream ERK pathway.
Assuntos
Quilomícrons , Dieta Hiperlipídica , Sistema de Sinalização das MAP Quinases , Animais , Masculino , Camundongos , Aciltransferases/metabolismo , Aciltransferases/genética , Antígenos CD36/metabolismo , Antígenos CD36/genética , Quilomícrons/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/metabolismo , Absorção Intestinal/efeitos dos fármacos , Ácidos Linoleicos Conjugados/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Obesity leads to a decline in the exercise capacity of skeletal muscle, thereby reducing mobility and promoting obesity-associated health risks. Dietary intervention has been shown to be an important measure to regulate skeletal muscle function, and previous studies have demonstrated the beneficial effects of docosahexaenoic acid (DHA; 22:6 ω-3) on skeletal muscle function. At the molecular level, DHA and its metabolites were shown to be extensively involved in regulating epigenetic modifications, including DNA methylation, histone modifications, and small non-coding microRNAs. However, whether and how epigenetic modification of mRNA such as N6-methyladenosine (m6A) mediates DHA regulation of skeletal muscle function remains unknown. Here, we analyze the regulatory effect of DHA on skeletal muscle function and explore the involvement of m6A mRNA modifications in mediating such regulation. RESULTS: DHA supplement prevented HFD-induced decline in exercise capacity and conversion of muscle fiber types from slow to fast in mice. DHA-treated myoblasts display increased mitochondrial biogenesis, while slow muscle fiber formation was promoted through DHA-induced expression of PGC1α. Further analysis of the associated molecular mechanism revealed that DHA enhanced expression of the fat mass and obesity-associated gene (FTO), leading to reduced m6A levels of DNA damage-induced transcript 4 (Ddit4). Ddit4 mRNA with lower m6A marks could not be recognized and bound by the cytoplasmic m6A reader YTH domain family 2 (YTHDF2), thereby blocking the decay of Ddit4 mRNA. Accumulated Ddit4 mRNA levels accelerated its protein translation, and the consequential increased DDIT4 protein abundance promoted the expression of PGC1α, which finally elevated mitochondria biogenesis and slow muscle fiber formation. CONCLUSIONS: DHA promotes mitochondrial biogenesis and skeletal muscle fiber remodeling via FTO/m6A/DDIT4/PGC1α signaling, protecting against obesity-induced decline in skeletal muscle function.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato , Ácidos Docosa-Hexaenoicos , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Animais , Dieta , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Obesidade , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/farmacologia , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND/OBJECTIVE: N6-methyladenosine (m6A) modification of mRNA plays a role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, plays a critical role in anti-obesity and anti-adipogenesis. METHODS: High-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HPLC-QqQ-MS/MS) was performed to determine the m6A levels in 3T3-L1 preadipocytes. The effects of EGCG on the m6A levels in specific genes were determined by methylated RNA immunoprecipitation coupled with quantitative real-time PCR (meRIP-qPCR). Several adipogenesis makers and cell cycle genes were analyzed by quantitative real-time PCR (qPCR) and western blotting. Lipid accumulation was evaluated by oil red O staining. All measurements were performed at least for three times. RESULTS: Here we showed that EGCG inhibited adipogenesis by blocking the mitotic clonal expansion (MCE) at the early stage of adipocyte differentiation. Exposing 3T3-L1 cells to EGCG reduced the expression of fat mass and obesity-associated (FTO) protein, an m6A demethylase, which led to increased overall levels of RNA m6A methylation. Cyclin A2 (CCNA2) and cyclin dependent kinase 2 (CDK2) play vital roles in MCE. The m6A levels of CCNA2 and CDK2 mRNA were dramatically enhanced by EGCG. Interestingly, EGCG increased the expression of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), which recognized and decayed methylated mRNAs, resulting in decreased protein levels of CCNA2 and CDK2. As a result, MCE was blocked and adipogenesis was inhibited. FTO overexpression and YTHDF2 knockdown in 3T3-L1 cells significantly increased CCNA2 and CDK2 protein levels and ameliorated the EGCG-induced adipogenesis inhibition. Thus, m6A-dependent CCNA2 and CDK2 expressions mediated by FTO and YTHDF2 contributed to EGCG-induced adipogenesis inhibition. CONCLUSION: Our findings provide mechanistic insights into how m6A is involved in the EGCG regulation of adipogenesis and shed light on its anti-obesity effect.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Fármacos Antiobesidade/farmacologia , Catequina/análogos & derivados , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células 3T3-L1/citologia , Adipócitos/citologia , Dioxigenase FTO Dependente de alfa-Cetoglutarato/deficiência , Animais , Catequina/farmacologia , Modelos Animais de Doenças , Camundongos , RNA Mensageiro/química , RNA Mensageiro/genética , Chá/químicaRESUMO
BACKGROUND: Shen-Fu decoction is a traditional Chinese medicine prescription with a 3:2 ratio of Radix Ginseng and Fuzi (Radix Aconiti lateralis praeparata). Ginsenosides and alkaloids are considered to be the main active components of Shen-Fu decoction. However, no analytical methods have been used to quantitatively analyse both components in Shen-Fu decoction simultaneously. RESULTS: We successfully developed a rapid resolution liquid chromatography coupled with tandem mass spectrometry (RRLC-MS/MS) method for the simultaneous analysis of seven ginsenosides and three aconitum alkaloids in Shen-Fu decoction, the decoction of Radix ginseng and Fuzi (Radix Aconiti lateralis praeparata). Chromatogrpahic separation by RPLC was achieved using a reversed-phase column and a water/acetonitrile mobile phase, containing 0.05% formic acid and using a gradient system. The method was optimized to allow for simultaneous analysis of all analytes in 11minutes without the need for baseline resolution of the components. Furthermore, the separation demonstrated good linearity (r > 0.9882), repeatability (RSD < 7.01%), intra- and inter-day precisions (RSD < 5.06%) and high yields of recovery (91.13-111.97%) for ten major constituents, namely ginsenoside-Re, Rg1, Rb1, Rc, Rb2, Rd, Rf, aconitine, hypacoitine and mesaconitine. CONCLUSIONS: The developed method could be used as a rapid and reliable approach for assessment of the quantity of the major constituents in Shen-Fu decoction.