LncRNA DLEU2 regulates sirtuins and mitochondrial respiratory chain complex IV: a novel pathway in obesity and offspring's health.

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a rapidly expanding area of interest in chronic diseases. They are mostly unknown for roles in metabolic regulation. Sirtuins, an epigenetic modulator class, regulate metabolic pathways. However, how sirtuins are regulated via lncRNA is unknown. We hypothesized that a high-fat high-fructose diet (HFD-HF) during pregnancy would increase the risk for obesity via lncRNA-Sirtuin pathways. METHODS: Female C57Bl/6 mice (F0) were fed either chow diet (CD) or HFD-HF for 6 weeks till birth. The pups (F1) were fed either CD or HFD-HF for 20 weeks. Expression of Dleu2, sirtuins, mitochondrial respiratory complexes, and oxidative stress were investigated in the F1 livers. Fasting blood glucose, insulin sensitivity, glucose tolerance, body and tissues weight were measured. A mechanistic interaction was then carried out using a DLEU2 knockdown experiment in the HepG2 cell. RESULTS: Dleu2 and sirtuins were both significantly decreased in the livers of HFD-HF fed male F1 whose mothers were either fed CD or HFD-HF during reproductive and pregnancy windows. Confirming this connection, upon silencing DLEU2, transcription levels of SIRT1 through 6 and translational levels of SIRT1, 3, 5, and 6 were significantly downregulated. Knockdown of DLEU2 significantly decreased the protein level of cytochrome-c oxidase (complex IV, MTCO1) without altering other mitochondrial complexes, decreased mitochondrial membrane potential, decreased ATP, and increased reactive oxygen species. Interestingly, in F1 livers, the protein level of MTCO1 was also significantly decreased under an HFD-HF diet or even under chow diet if the mother was exposed to HFD-HF. CONCLUSION: Our findings reveal for the first time that one lncRNA can regulate sirtuins and a specific mitochondrial complex. Furthermore, diet or maternal diet can modulate Dleu2 and its downstream regulators in offspring, suggesting a potential role of DLEU2 in metabolic disorders over one or more generations.

Screening for alternative splicing of lncRNA Dleu2 in the mouse liver cell line AML-12.

The long non coding RNA deleted in leukemia 2 gene (Dleu2) has recently been demonstrated to be an active player in the progression of several types of cancer, including hepatocellular carcinoma. Dleu2 may serve a role in modulating the downstream effects-mediated by alternative splicing of its multiple exons. However, the proportional expression of these alternative splicing populations of the Dleu2 exons is currently unknown. To determine how Dleu2 could be affected by alternative splicing, a series of alternative splicing primer sets were designed to investigate which transcripts were preferentially activated when Dleu2 was targeted for downregulation or upregulation. A specific Dleu2 small interfering RNA that targeted an exon upstream of the tumor suppressor microRNA site significantly knocked down Dleu2 expression across all the primer sets used, which targeted 13 different alternative splicing transcripts over 5 different promoter sites in the mouse liver cell line, AML-12. Similarly, 50 µM Resveratrol led to significant upregulation of Dleu2 in 11 alternative splicing transcripts. These results show that Dleu2 is capable of successful modulation across alternative splicing transcripts that can be screened, and also that Resveratrol can be a potential nutraceutical, which may potentially lead to novel approaches in the use of lncRNA Dleu2 for diagnostics and regulation.