Regulation of Na/K-ATPase expression by cholesterol: isoform specificity and the molecular mechanism

We have reported that the reduction in plasma membrane cholesterol could decrease cellular Na/K-ATPase α1-expression through a Src-dependent pathway. However, it is unclear whether cholesterol could regulate other Na/K-ATPase α-isoforms and the molecular mechanisms of this regulation are not fully understood. Here we used cells expressing different Na/K-ATPase α isoforms and found that membrane cholesterol reduction by U18666A decreased expression of the α1-isoform but not the α2- or α3-isoform. Imaging analyses showed the cellular redistribution of α1 and α3 but not α2. Moreover, U18666A led to redistribution of α1 to late endosomes/lysosomes, while the proteasome inhibitor blocked α1-reduction by U18666A. These results suggest that the regulation of the Na/K-ATPase α-subunit by cholesterol is isoform specific and α1 is unique in this regulation through the endocytosis-proteasome pathway. Mechanistically, loss-of-Src binding mutation of A425P in α1 lost its capacity for regulation by cholesterol. Meanwhile, gain-of-Src binding mutations in α2 partially restored the regulation. Furthermore, through studies in caveolin-1 knockdown cells, as well as subcellular distribution studies in cell lines with different α-isoforms, we found that Na/K-ATPase, Src, and caveolin-1 worked together for the cholesterol regulation. Taken together, these new findings reveal that the putative Src-binding domain and the intact Na/K-ATPase/Src/caveolin-1 complex are indispensable for the isoform-specific regulation of Na/K-ATPase by cholesterol.

The Effect of Salam Leaf Extract (Syzygium polianthum) Against Cholesterol Regulation in Hypercholesterolemic Model White Rats

Cholesterol is an important precursor that plays a role in the production andregulation of various sterol group compounds, especially steroid hormonecompounds, androgen hormone compounds, cortisol compounds and estrogencompounds. This herb is known to have the effect of improving glucose regulationby increasing glucose intake into cells and tissues. Its ability to improve cell andtissue metabolism is mediated by the content of secondary metabolite compounds.This ability is believed to have the potential to improve cholesterol regulation. Bayleaf extract is effective in lowering cholesterol levels through regulation of Acetyl CoAproduction.

Abstract 472: Liver X Receptor Ligands Suppress Intestinal Soat2 and Reduce Cholesterol in Mice Fed a High Cholesterol Diet

The nuclear receptors, liver X receptor-alpha and -beta (LXRa and LXRb), were originally identified as orphan members of the nuclear receptor (NR) superfamily that function as heterodimers with the retinoid X receptor (RXR). Both LXRa and LXRb play important roles in cholesterol homeostasis and lipid metabolism and have been implicated in the pathology of several diseases including atherosclerosis, cancer, and obesity. Detailed examination of mice deficient in LXRa have revealed significant information regarding its role in regulating lipid metabolism while pharmacological roles of LXR in metabolism and inflammation have been identified using synthetic agonists. We have identified novel synthetic LXR Inverse Agonists that display the ability to suppress lipogenesis and significantly improve the phenotype of fatty liver diseases in several mouse models. During our previous investigation of LXR inverse agonists and fatty liver, we observed a substantial decrease in LDL and total cholesterol, as well as a significant decrease in soat2 gene expression. Recent basic and clinical studies indicate that soat2 may be a potential therapeutic target for cholesterol regulation and atherosclerosis treatment. Based on our observations, we investigated the effects of tissue-selective LXR inverse agonists on cholesterol regulation with greater intensity. We hypothesized that soat2 may be a novel target of LXR and that selectively suppressing LXR in the liver and/or intestine may lead to lowered cholesterol levels in hyperlipidemic models (n = 12). While our research is currently ongoing, we have validated soat2 as a potential LXR target gene by ChIP in hepatocytes, RNA-Seq experiments, fecal cholesterol excretion experiments, as well as expression assays in LXRa/b double knockout mouse tissues (n = 3). Additional studies in LDLr KO mice indicate that selectively targeting LXR in the gut with inverse agonists significantly decrease total and LDL cholesterol (n=12). We believe that this is an innovative method of targeting hypercholesterolemia and may lead to a new class of atherosclerosis therapeutics.

CRISPR-Cas12a delivery by DNA-mediated bioresponsive editing for cholesterol regulation

CRISPR-Cas12a represents an efficient tool for genome editing in addition to the extensively investigated CRISPR-Cas9. However, development of efficient nonviral delivery system for CRISPR-Cas12a remains challenging. Here, we demonstrate a DNA nanoclew (NC)–based carrier for delivery of Cas12a/CRISPR RNA (crRNA) ribonucleoprotein (RNP) toward regulating serum cholesterol levels. The DNA NC could efficiently load the Cas12a/crRNA RNP through complementation between the DNA NC and the crRNA. Addition of a cationic polymer layer condensed the DNA-templated core and allowed further coating of a charge reversal polymer layer, which makes the assembly negatively charged under a physiological pH but reverts to positive charge under an acidic environment. When Pcsk9 was selected as the target gene because of its important role in regulating the level of serum cholesterol, efficient Pcsk9 disruption was observed in vivo (~48%), significantly reducing the expression of PCSK9 and gaining the therapeutic benefit of cholesterol control (~45% of cholesterol reduction).