Purification, full-length sequencing and genomic origin mapping of eccDNA.

Extrachromosomal circular DNA (eccDNA) was discovered more than half a century ago. However, its biogenesis and function have just begun to be elucidated. One hurdle that has prevented our understanding of eccDNA is the difficulty in obtaining pure eccDNA from cells. The current eccDNA purification methods mainly rely on depleting linear DNAs by exonuclease digestion after obtaining crude circles by alkaline lysis. Owing to eccDNA's low abundance and heterogeneous size, the current purification methods are not efficient in obtaining pure eccDNA. Here we describe a new three-step eccDNA purification (3SEP) procedure that adds a step to recover circular DNA, but not linear DNA that escape from the exonuclease digestion, whereby 3SEP results in eccDNA preparations with high purity and reproducibility. Additionally, we developed a full-length eccDNA sequencing technique by combining rolling-circle amplification with Nanopore sequencing. Accordingly, we developed a full-length eccDNA caller (Flec) to call the consensus sequence of multiple tandem copies of eccDNA contained within the debranched rolling-circle amplification product and map the consensus to its genomic origin. Collectively, our protocol will facilitate eccDNA identification and characterization, and has the potential for diagnostic and clinical applications. For a well-trained molecular biologist, it takes ~1-2 d to purify eccDNAs, another 5-6 d to carry out Nanopore library preparation and sequencing, and 1-5 d for an experienced bioinformatic scientist to analyze the data.

eccDNAs are apoptotic products with high innate immunostimulatory activity.

Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species1,2. However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response.

Inhibition of CK1ε potentiates the therapeutic efficacy of CDK4/6 inhibitor in breast cancer.

Although inhibitors targeting CDK4/6 kinases (CDK4/6i) have shown promising clinical prospect in treating ER+/HER2- breast cancers, acquired drug resistance is frequently observed and mechanistic knowledge is needed to harness their full clinical potential. Here, we report that inhibition of CDK4/6 promotes ßTrCP1-mediated ubiquitination and proteasomal degradation of RB1, and facilitates SP1-mediated CDK6 transcriptional activation. Intriguingly, suppression of CK1ε not only efficiently prevents RB1 from degradation, but also prevents CDK4/6i-induced CDK6 upregulation by modulating SP1 protein stability, thereby enhancing CDK4/6i efficacy and overcoming resistance to CDK4/6i in vitro. Using xenograft and PDX models, we further demonstrate that combined inhibition of CK1ε and CDK4/6 results in marked suppression of tumor growth in vivo. Altogether, these results uncover the molecular mechanisms by which CDK4/6i treatment alters RB1 and CDK6 protein abundance, thereby driving the acquisition of CDK4/6i resistance. Importantly, we identify CK1ε as an effective target for potentiating the therapeutic efficacy of CDK4/6 inhibitors.

Hemin Improves Insulin Sensitivity and Lipid Metabolism in Cultured Hepatocytes and Mice Fed a High-Fat Diet.

Hemin is a breakdown product of hemoglobin. It has been reported that the injection of hemin improves lipid metabolism and insulin sensitivity in various genetic models. However, the effect of hemin supplementation in food on lipid metabolism and insulin sensitivity is still unclear, and whether hemin directly affects cellular insulin sensitivity is yet to be elucidated. Here we show that hemin enhances insulin-induced phosphorylation of insulin receptors, Akt, Gsk3ß, FoxO1 and cytoplasmic translocation of FoxO1 in cultured primary hepatocytes under insulin-resistant conditions. Furthermore, hemin diminishes the accumulation of triglyceride and increases in free fatty acid content in primary hepatocytes induced by palmitate. Oral administration of hemin decreases body weight, energy intake, blood glucose and triglyceride levels, and improves insulin and glucose tolerance as well as hepatic insulin signaling and hepatic steatosis in male mice fed a high-fat diet. In addition, hemin treatment decreases the mRNA and protein levels of some hepatic genes involved in lipogenic regulation, fatty acid synthesis and storage, and increases the mRNA level and enzyme activity of CPT1 involved in fatty acid oxidation. These data demonstrate that hemin can improve lipid metabolism and insulin sensitivity in both cultured hepatocytes and mice fed a high-fat diet, and show the potential beneficial effects of hemin from food on lipid and glucose metabolism.

Down-regulation of Risa improves insulin sensitivity by enhancing autophagy.

It has been reported that some small noncoding RNAs are involved in the regulation of insulin sensitivity. However, whether long noncoding RNAs also participate in the regulation of insulin sensitivity is still largely unknown. We identified and characterized a long noncoding RNA, regulator of insulin sensitivity and autophagy (Risa), which is a poly(A)(+) cytoplasmic RNA. Overexpression of Risa in mouse primary hepatocytes or C2C12 myotubes attenuated insulin-stimulated phosphorylation of insulin receptor, Akt, and Gsk3ß, and knockdown of Risa alleviated insulin resistance. Further studies showed that overexpression of Risa in hepatocytes or myotubes decreased autophagy, and knockdown of Risa up-regulated autophagy. Moreover, knockdown of Atg7 or -5 significantly inhibited the effect of knockdown of Risa on insulin resistance, suggesting that knockdown of Risa alleviated insulin resistance via enhancing autophagy. In addition, tail vein injection of adenovirus to knock down Risa enhanced insulin sensitivity and hepatic autophagy in both C57BL/6 and ob/ob mice. Taken together, the data demonstrate that Risa regulates insulin sensitivity by affecting autophagy and suggest that Risa is a potential target for treating insulin-resistance-related diseases.-Wang, Y., Hu, Y., Sun, C., Zhuo, S., He, Z., Wang, H., Yan, M., Liu, J., Luan, Y., Dai, C., Yang, Y., Huang, R., Zhou, B., Zhang, F., Zhai, Q. Down-regulation of Risa improves insulin sensitivity by enhancing autophagy.

CLOCK and BMAL1 Regulate Muscle Insulin Sensitivity via SIRT1 in Male Mice.

Circadian misalignment induces insulin resistance in both human and animal models, and skeletal muscle is the largest organ response to insulin. However, how circadian clock regulates muscle insulin sensitivity and the underlying molecular mechanisms are still largely unknown. Here we show circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1, two core circadian transcription factors, are down-regulated in insulin-resistant C2C12 myotubes and mouse skeletal muscle. Furthermore, insulin signaling is attenuated in the skeletal muscle of Clock(Δ19/Δ19) mice, and knockdown of CLOCK or BMAL1 by small interfering RNAs induces insulin resistance in C2C12 myotubes. Consistently, ectopic expression of CLOCK and BMAL1 improves insulin sensitivity in C2C12 myotubes. Moreover, CLOCK and BMAL1 regulate the expression of sirtuin 1 (SIRT1), an important regulator of insulin sensitivity, in C2C12 myotubes and mouse skeletal muscle, and two E-box elements in Sirt1 promoter are responsible for its CLOCK- and BMAL1-dependent transcription in muscle cells. Further studies show that CLOCK and BMAL1 regulate muscle insulin sensitivity through SIRT1. In addition, we find that BMAL1 and SIRT1 are decreased in the muscle of mice maintained in constant darkness, and resveratrol supplementation activates SIRT1 and improves insulin sensitivity. All these data demonstrate that CLOCK and BMAL1 regulate muscle insulin sensitivity via SIRT1, and activation of SIRT1 might be a potential valuable strategy to attenuate muscle insulin resistance related to circadian misalignment.

CLOCK/BMAL1 regulates circadian change of mouse hepatic insulin sensitivity by SIRT1.

UNLABELLED: The protein deacetylase, sirtuin 1 (SIRT1), involved in regulating hepatic insulin sensitivity, shows circadian oscillation and regulates the circadian clock. Recent studies show that circadian misalignment leads to insulin resistance (IR); however, the underlying mechanisms are largely unknown. Here, we show that CLOCK and brain and muscle ARNT-like protein 1 (BMAL1), two core circadian transcription factors, are correlated with hepatic insulin sensitivity. Knockdown of CLOCK or BMAL1 induces hepatic IR, whereas their ectopic expression attenuates hepatic IR. Moreover, circadian change of insulin sensitivity is impaired in Clock mutant, liver-specific Bmal1 knockout (KO) or Sirt1 KO mice, and CLOCK and BMAL1 are required for hepatic circadian expression of SIRT1. Further studies show that CLOCK/BMAL1 binds to the SIRT1 promoter to enhance its expression and regulates hepatic insulin sensitivity by SIRT1. In addition, constant darkness-induced circadian misalignment in mice decreases hepatic BMAL1 and SIRT1 levels and induces IR, which can be dramatically reversed by resveratrol. CONCLUSION: These findings offer new insights for coordination of the circadian clock and metabolism in hepatocytes by circadian regulation of hepatic insulin sensitivity via CLOCK/BMAL1-dependent SIRT1 expression and provide a potential application of resveratrol for combating circadian misalignment-induced metabolic disorders.

A high-throughput quantitative approach reveals more small RNA modifications in mouse liver and their correlation with diabetes.

Studies of RNA modification are usually focused on tRNA. However the modification of other small RNAs, including 5.8S rRNA, 5S rRNA, and small RNA sized at 10-60 nt, is still largely unknown. In this study, we established an efficient method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously identify and quantify more than 40 different types of nucleosides in small RNAs. With this method, we revealed 23 modified nucleosides of tRNA from mouse liver, and 6 of them were observed for the first time in eukaryotic tRNA. Moreover, 5 and 4 modified nucleosides were detected for the first time in eukaryotic 5.8S and 5S rRNA, respectively, and 22 modified nucleosides were identified in the small RNAs sized at 30-60 or 10-30 nt. Interestingly, two groups of 5S rRNA peaks were observed when analyzed by HPLC, and the abundance of modified nucleosides is significantly different between the two groups of peaks. Further studies show that multiple modifications in small RNA from diabetic mouse liver are significantly increased or decreased. Taken together, our data revealed more modified nucleosides in various small RNAs and showed the correlation of small RNA modifications with diabetes. These results provide new insights to the role of modifications of small RNAs in their stability, biological functions, and correlation with diseases.

Amyloid-ß induces hepatic insulin resistance in vivo via JAK2.

Amyloid-ß (Aß), a natural product of cell metabolism, plays a key role in the pathogenesis of Alzheimer's disease (AD). Epidemiological studies indicate patients with AD have an increased risk of developing type 2 diabetes mellitus (T2DM). Aß can induce insulin resistance in cultured hepatocytes by activating the JAK2/STAT3/SOCS-1 signaling pathway. Amyloid precursor protein and presenilin 1 double-transgenic AD mouse models with increased circulating Aß level show impaired glucose/insulin tolerance and hepatic insulin resistance. However, whether Aß induces hepatic insulin resistance in vivo is still unclear. Here we show C57BL/6J mice intraperitoneally injected with Aß42 exhibit increased fasting blood glucose level, impaired insulin tolerance, and hepatic insulin signaling. Moreover, the APPswe/PSEN1dE9 AD model mice intraperitoneally injected with anti-Aß neutralizing antibodies show decreased fasting blood glucose level and improved insulin sensitivity. Injection of Aß42 activates hepatic JAK2/STAT3/SOCS-1 signaling, and neutralization of Aß in APPswe/PSEN1dE9 mice inhibits liver JAK2/STAT3/SOCS-1 signaling. Furthermore, knockdown of hepatic JAK2 by tail vein injection of adenovirus inhibits JAK2/STAT3/SOCS-1 signaling and improves glucose/insulin tolerance and hepatic insulin sensitivity in APPswe/PSEN1dE9 mice. Our results demonstrate that Aß induces hepatic insulin resistance in vivo via JAK2, suggesting that inhibition of Aß signaling is a new strategy toward resolving insulin resistance and T2DM.