Our study unveils an H19/PTBP1/sterol regulatory element-binding protein 1 feedforward amplifying signaling pathway to exacerbate the development of fatty liver. (Hepatology 2018;67:1768-1783).
The development of high-throughput single-cell RNA sequencing (scRNA-seq) has enabled access to information about gene expression in individual cells and insights into new biological areas. Although the interest in scRNA-seq has rapidly grown in recent years, the existing methods are plagued by many challenges when performing scRNA-seq on multiple samples. To simultaneously analyze multiple samples with scRNA-seq, we developed a universal sample barcoding method through transient transfection with short barcode oligonucleotides. By conducting a species-mixing experiment, we have validated the accuracy of our method and confirmed the ability to identify multiplets and negatives. Samples from a 48-plex drug treatment experiment were pooled and analyzed by a single run of Drop-Seq. This revealed unique transcriptome responses for each drug and target-specific gene expression signatures at the single-cell level. Our cost-effective method is widely applicable for the single-cell profiling of multiple experimental conditions, enabling the widespread adoption of scRNA-seq for various applications.
Objective
Proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds the low density lipoprotein (LDL) receptor and targets it for degradation, has emerged as an important regulator of serum cholesterol levels and cardiovascular disease risk. Although much work is currently focused on developing therapies for inhibiting PCSK9, the endogenous regulation of PCSK9, particularly by insulin, remains unclear. The objective of these studies was to determine the effects of insulin on PCSK9 in vitro and in vivo.
Approach and Results
Using rat hepatoma cells and primary rat hepatocytes, we found that insulin increased PCSK9 expression and increased LDL receptor degradation in a PCSK9-dependent manner. In parallel, hepatic Pcsk9 mRNA and plasma PCSK9 protein levels were reduced by 55-75% in mice with liver-specific knockout of the insulin receptor; 75-88% in mice made insulin deficient with streptozotocin; and 65% in ob/ob mice treated with antisense oligonucleotides against the insulin receptor. However, antisense olignonucleotide mediated knockdown of insulin receptor in lean, wildtype mice had little effect. In addition, we found that fasting was able to reduce PCSK9 expression by 80% even in mice that lack hepatic insulin signaling.
Conclusions
Taken together, these data indicate that though insulin induces PCSK9 expression, it is not the sole or even dominant regulator of PCSK9 under all conditions.
Cell growth and proliferation are tightly coupled to metabolism, and dissecting the signaling molecules which link these processes is an important step towards understanding development, regeneration and cancer. The transcriptional regulator Yes-associated protein 1 (YAP) is a key regulator of liver size, development and function. We now show that YAP can also suppress gluconeogenic gene expression. Yap deletion in primary hepatocytes potentiates the gluconeogenic gene response to glucagon and dexamethasone, whereas constitutively active YAP suppresses it. The effects of YAP are mediated by the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1α). YAP inhibits the ability of PGC1α to bind to and activate transcription from the promoters of its gluconeogenic targets and the effects of YAP are blunted upon knockdown of PGC1α. In vivo, constitutively active YAP lowers plasma glucose levels and increases liver size. YAP therefore appears to reprogram cellular metabolism, diverting substrates away from the energy consuming process of gluconeogenesis, and towards the anabolic process of growth.
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