The absorption of dietary lipids takes place mainly in the proximal small intestine. In the intestinal lumen, cholesterol is emulsified by bile salts and incorporated along with fatty acids into micelles (116). After cholesterol enters enterocytes, it moves to the ER, where it can be esterified by ACAT2 (117, 118) and incorporated into chylomicrons for systemic absorption. Niemann-Pick C1–like 1 (NPC1L1) has a critical role in the initial phase of cholesterol uptake into the enterocyte PM (119). However, the fate of cholesterol downstream of its uptake into PM of enterocytes has remained unclear. Recent work has described a role for Aster-B and Aster-C in mediating the nonvesicular trafficking of diet-derived cholesterol from PM to ER in the small intestine.

Gramd1b and Gramd1c are highly expressed in differentiated enterocytes, while Gramd1a is prevalent in other intestinal cell types, including crypt stem cells and T cells (120). Intestinal deletion of Aster-B and -C results in lower uptake of diet-derived cholesterol into enterocytes and into the circulation, and attenuates diet-induced hypercholesterolemia (120). Importantly, the absorption of fatty acids and glucose is preserved in the absence of Asters, indicating a specific role in cholesterol handling. Mice lacking intestinal Asters maintain the capacity to assemble chylomicrons, but their chylomicrons have reduced cholesterol content. Studies in intestinal enteroids showed that Aster ablation impairs the trafficking of cholesterol from the apical PM to the ER. As a result, the pool of accessible cholesterol at the membrane of enterocytes expands, ER cholesterol is depleted, and the SREBP2 pathway for cholesterol synthesis is induced.

Functional NPC1L1 is essential to make dietary cholesterol available for transfer to the ER and for its subsequent incorporation into chylomicrons for absorption. Structural biology studies have helped elucidate the mechanism by which NPC1L1 mediates cholesterol entry and membrane deposition in enterocytes (121–123). Cryogenic electron microscopy experiments showed that the N-terminal domain of NPC1L1 possesses a cavity that can bind cholesterol, and that the binding of cholesterol makes the domain mobile. Rotation of the domain forms a tunnel that allows cholesterol to enter the membrane. Cell biology studies have shown that NPC1L1 is internalized via clathrin-dependent endocytosis when enterocytes are exposed to dietary cholesterol (61). The clathrin adaptor Numb binds the C-terminal domain of NPC1L1 and recruits AP2 and clathrin (124). NPC1L1 colocalizes with Rab11 after oral cholesterol administration (125), indicating that NPC1L1 endocytic vesicles are incorporated into the endocytic recycling compartment (ERC). More recently, LIM domain and actin-binding 1 (LIMA1) has been identified as a regulator of intestinal absorption owing to its ability to facilitate NPC1L1 recycling (126). These findings support the importance of NPC1L1 recycling for the absorption of dietary cholesterol. However, whether cholesterol reaches the ER via NPC1L1-mediated ERC has not been established.

Our work suggests that Asters work in concert with NPC1L1 (Figure 4) to allow dietary cholesterol to rapidly reach the enterocyte ER. The saturation of the apical PM with cholesterol by NPC1L1 is necessary to recruit Aster-B and Aster-C to the apical PM of enterocytes (120). Genetic deletion of NPC1L1 or inhibition by ezetimibe blocks Aster recruitment to the PM by cholesterol in vivo, positioning NPC1L1 upstream of the Aster pathway. Genetic deletion of Asters impairs cholesterol movement to the ER, even when NPC1L1 is present. Thus, NPC1L1 alone cannot fully explain cholesterol movement to the ER in enterocytes. This model for cooperation between NPC1L1 and Asters does not rule out that some cholesterol may be incorporated into endosomes and ERC along with NPC1L1, and from there undergo vesicular transfer to the ER (Figure 4). Additionally, it seems likely that other proteins participate in nonvesicular or vesicular transport in enterocytes, as cholesterol absorption is not completely abrogated in Aster-knockout models.

Aster-mediated trafficking of cholesterol in enterocytes. NPC1L1 controls the first step of the process of dietary cholesterol absorption, by gating diet-derived cholesterol uptake by enterocytes. NPC1L1 is present at the apical membrane of the enterocytes when the concentration of cholesterol is low, and it allows the entry of cholesterol and deposition in the lipid bilayer. This expands the pool of accessible cholesterol at the apical membrane and engages Aster-B and Aster-C. NPC1L1 is internalized in clathrin-coated vesicles, recycled in the endocytic recycling compartment (ERC), and redirected to the membrane. The movement of cholesterol from PM to ER allows cholesterol esterification and incorporation onto chylomicrons for systemic absorption. It has been speculated that Asters may also be recruited to cholesterol-enriched ERC, but this requires further study. Aster-mediated trafficking of cholesterol to the ER decreases the level of cholesterol in the PM, thus favoring NPC1L1 recycling.

Pharmacological inhibition of NPC1L1 by the hypocholesterolemic drug ezetimibe is highly effective at blocking cholesterol absorption. Interestingly, Aster-mediated nonvesicular trafficking in intestine is also pharmacologically targetable. The small-molecule inhibitor AI-3d mimics the effects of Aster genetic deletion on cholesterol movement in enterocytes (120). In murine and human enteroids, treatment with AI-3d expands the pool of accessible cholesterol in the PM. Moreover, oral administration of AI-3d to mice reduces cholesterol trafficking to the ER and cholesterol absorption. The Aster pathway may offer an additional opportunity for pharmacological manipulation of dietary cholesterol uptake.