Establishment of a Drosophila model of intestinal sterol absorption and trafficking
Despite diet being a major source of cholesterol in Western culture, the absorption and downstream trafficking of cholesterol remain poorly understood processes. In humans, proteins of the NPC1 family are essential for these processes. Niemann-Pick Type C1 (NPC1) promotes intracellular sterol trafficking in all tissues, while Niemann-Pick Type C1-Like 1 (NPC1L1) performs as an essential early function in the absorption pathway. However, the specific mechanism by which these proteins function remains unidentified. This dissertation details my efforts to establish a model of dietary sterol absorption and trafficking using the fruit fly Drosophila melanogaster through the study of fly homologs of the NPC1 family. Previous work identified the protein NPC1a as the fly homolog of NPC1, while my work primarily focused on studying the functions of the second fly NPC1 family member, NPC1b. Animals lacking NPC1b protein die during larval development. Based on the known roles of the human NPC1 family, I hypothesized that NPC1b functions either specifically in the absorption of dietary sterols or in general intracellular trafficking of sterols. A novel dietary sterol absorption assay revealed that NPC1b mutants have dramatically reduced rates of sterol absorption. I found that the essential function of NPC1b is restricted to the midgut, and NPC1b mutant midgut tissues are dramatically devoid of free sterols, indicating that NPC1b must perform an early role in sterol absorption. Together, my results indicate that the essential role for NPC1b lies in the process of dietary sterol absorption. Curiously, in the absence of both NPC1b and NPC1a, dietary sterol absorption rates are comparable to wild-type levels, suggesting that there is a secondary sterol absorption pathway normally inhibited by NPC1a. I found that this secondary pathway is regulated by 20-hydroxyecdysone. These findings suggest that the NPC1 family members are both involved in dietary sterol absorption. A major role of NPC1b is to facilitate absorption of dietary sterols, while NPC1a acts through ecdysone to inhibit a secondary absorption pathway. These findings provide new insights into dietary sterol absorption and trafficking and will allow us to further study the absorption pathway and functions of the NPC1 family.
- Genetics