Adipocyte HSL is required for maintaining circulating vitamin A and RBP4 levels during fasting.

Vitamin A (retinol) is distributed via the blood bound to its specific carrier protein, retinol-binding protein 4 (RBP4). Retinol-loaded RBP4 is secreted into the circulation exclusively from hepatocytes, thereby mobilizing hepatic retinoid stores that represent the major vitamin A reserves in the body. The relevance of extrahepatic retinoid stores for circulating retinol and RBP4 levels that are usually kept within narrow physiological limits is unknown. Here, we show that fasting affects retinoid mobilization in a tissue-specific manner, and that hormone-sensitive lipase (HSL) in adipose tissue is required to maintain serum concentrations of retinol and RBP4 during fasting in mice. We found that extracellular retinol-free apo-RBP4 induces retinol release by adipocytes in an HSL-dependent manner. Consistently, global or adipocyte-specific HSL deficiency leads to an accumulation of retinoids in adipose tissue and a drop of serum retinol and RBP4 during fasting, which affects retinoid-responsive gene expression in eye and kidney and lowers renal retinoid content. These findings establish a novel crosstalk between liver and adipose tissue retinoid stores for the maintenance of systemic vitamin A homeostasis during fasting.

Knockout of ABCC1 in NCI-H441 cells reveals CF to be a suboptimal substrate to study MRP1 activity in organotypic in vitro models.

Multidrug resistance-associated protein 1 (MRP1/ABCC1) is an efflux transporter responsible for the extrusion of endogenous substances as well as xenobiotics and their respective metabolites. Its high expression levels in lung tissue imply a key role in pulmonary drug disposition. Moreover, its association with inflammatory lung diseases underline MRP1's relevance in drug development and precision-medicine. With the aim to develop a tool to better understand MRP1's role in drug disposition and lung disease, we generated an ABCC1-/- clone based on the human distal lung epithelial cell line NCI-H441 via a targeted CRISPR/Cas9 approach. Successful knockout (KO) of MRP1 was confirmed by qPCR, immunoblot and Sanger sequencing. To assess potential compensatory upregulation of transporters with a similar substrate recognition pattern as MRP1, expression levels of MRP2-9 as well as OAT1-4, 6, 7 and 10 were measured. Functional transporter activity was determined via release studies with two prodrug/substrate pairs, i.e. 5(6)-carboxyfluorescein (CF; formed from its diacetate prodrug) and S-(6-(7-methylpurinyl))glutathione (MPG; formed from its prodrug 6-bromo-7-methylpurine, BMP), respectively. Lastly, transepithelial electrical resistance (TEER) of monolayers of the KO clone were compared with wildtype (WT) NCI-H441 cells. Of eight initially generated clones, the M2 titled clone showed complete absence of mRNA and protein in accordance with the designed genome edit. In transport studies using the substrate CF, however, no differences between the KO clone and WT NCI-H441 cells were observed, whilst no differences in expression of potential compensatory transporters was noted. On the other hand, when using BMP/MPG, the release of MPG was reduced to 11.5% in the KO clone. Based on these results, CF appears to be a suboptimal probe for the study of MRP1 function, particularly in organotypic in vitro and ex vivo models. Our ABCC1-/- NCI-H441 clone further retained the ability to form electrically tight barriers, making it a useful model to study MRP1 function in vitro.