Dysregulated autophagy is associated with steatosis and non-alcoholic fatty liver disease (NAFLD), however the mechanisms connecting them remain poorly understand. Here, we show that co-administration of lovastatin and ezetimibe (L/E) significantly reverses hepatic triglyceride accumulation concomitant with an increase in SREBP-2 driven autophagy in mice fed a high-fat diet (HFD). We further show that the statin mediated increase in SREBP-2 directly activates expression of patatin-like phospholipase domain-containing enzyme 8 (PNPLA8) gene, and PNPLA8 associates with autophagosomes and is associated with a decrease in cellular triglyceride. Moreover, we show that over-expression of PNPLA8 dramatically decreases hepatic steatosis through increased autophagy in hepatocytes of HFD-fed mice. Live-cell imaging analyses also reveal that PNPLA8 dynamically interacts with LC3 and we suggest that the SREBP-2/PNPLA8 axis represents a novel regulatory mechanism for lipid homeostasis. These data provide a possible mechanism for the reported beneficial effects of statins for decreasing hepatic triglyceride levels in NAFLD patients.
Background The microRNAs (miRNAs) down-regulated in aged mouse skeletal muscle were mainly clustered within the delta-like homologue 1 and the type III iodothyronine deiodinase (Dlk1-Dio3) genomic region. Although clustered miRNAs are coexpressed and regulate multiple targets in a specific signalling pathway, the function of miRNAs in the Dlk1-Dio3 cluster in muscle aging is largely unknown. We aimed to ascertain whether these miRNAs play a common role to regulate age-related muscle atrophy. Methods To examine anti-atrophic effect of miRNAs, we individually transfected 42 miRNA mimics in fully differentiated myotubes and analysed their diameters. The luciferase reporter assay using target 3′ untranslated region (UTR) and RNA pull-down assay were employed to ascertain the target predicted by the TargetScan algorithm. To investigate the therapeutic potential of the miRNAs in vivo, we generated adeno-associated virus (AAV) serotype 9 expressing green fluorescent protein (GFP) (AAV9-GFP) bearing miR-376c-3p and infected it into the tibialis anterior muscle of old mice. We performed morphometric analysis and measured ex vivo isometric force using a force transducer. Human gluteus maximus muscle tissues (ages ranging from 25 to 80 years) were used to investigate expression levels of the conserved miRNAs in the Dlk1-Dio3 cluster. Results We found that the majority of miRNAs (33 out of 42 tested) in the cluster induced anti-atrophic phenotypes in fully differentiated myotubes with increasing their diameters. Eighteen of these miRNAs, eight of which are conserved in humans, harboured predicted binding sites in the 3′ UTR of muscle atrophy gene-1 (Atrogin-1) encoding a muscle-specific E3 ligase. Direct interactions were identified between these miRNAs and the 3′ UTR of Atrogin-1, leading to repression of Atrogin-1 and thereby induction of eIF3f protein content, in both human and mouse skeletal muscle cells. Intramuscular delivery of AAV9 expressing miR-376c-3p, one of the most effective miRNAs in myotube thickening, dramatically ameliorated skeletal muscle atrophy and improved muscle function, including isometric force, twitch force, and fatigue resistance in old mice. Consistent with our findings in mice, the expression of miRNAs in the cluster was significantly down-regulated in human muscle from individuals > 50 years old.
Skeletal muscle communicates with other organs via myokines, which are secreted by muscle during exercise and exert various effects. Despite much investigation of the exercise, the underlying molecular mechanisms are still not fully understood. Here, we applied an in vitro exercise model in which cultured C2C12 myotubes were subjected to electrical pulse stimulation (EPS), which mimics contracting muscle. Based on the significantly up- and down-regulated genes in EPS, we constructed an in silico model to predict exercise responses at the transcriptional level. The in silico model revealed similarities in the transcriptomes of the EPS and exercised animals. Comparative analysis of the EPS data and exercised mouse muscle identified putative biomarkers in exercise signaling pathways and enabled to discover novel exercise-induced myokines. Biochemical analysis of selected exercise signature genes in muscle from exercised mice showed that EPS mimics in vivo exercise, at least in part, at the transcriptional level. Consequently, we provide a novel myokine, Amphiregulin (AREG), up-regulated both in vitro and in vivo, that would be a potential target for exercise mimetics.
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