How Free Retinol Behaves Differently from RBP-Bound Retinol in

Berry et al. claimed that holo-retinol-binding protein (holo-RBP) binds to STRA6 to trigger insulin resistance but does not contribute to retinol uptake from holo-RBP (1). These claims, based on improperly performed assays, contradict many studies on STRA6 (2–10) and the findings that apo-RBP, not holo-RBP, is elevated in cases of obesity and type 2 diabetes (11, 12). The claims of Berry et al. also contradict two independent studies published in Molecular and Cellular Biology showing that RBP’s role in insu-lin resistance is independent of STRA6 and retinol (13, 14). Properly formed retinol/RBP complex, a cell type that is con-firmed to express the STRA6 protein, and the ability to distinguish true intracellular uptake from nonspecific binding (e.g., nonspe-cific sticking of [3H]retinol or [3H]retinol/RBP to plastic or cell surfaces) are prerequisites in STRA6-dependent vitamin A uptake assays. It has been well established from numerous studies, includ-ing crystallographic studies, that retinol forms a 1:1 complex with RBP. However, Berry et al. performed all [3H]retinol uptake as-says by mixing free [3H]retinol with unlabeled holo-RBP and ap-plying this mixture without removing free [3H]retinol. Because [3H]retinol/RBP was not properly formed and no free [3H]retinol was removed, this assay is a free [3H]retinol diffusion assay, not an assay of STRA6-mediated [3H]retinol uptake from the [3H]reti-nol/RBP complex (Fig. 1). It has been known since the 1970s that retinyl ester bound to lipoproteins is an RBP-independent mechanism of delivering vi-tamin A (15). Since RBP knockout mice have primarily vision defects under vitamin A-sufficient conditions (16, 17), it is no