Abstract 26: Identification of a Novel Gene, DENND5B, Associated With Plasma High Density Lipoprotein Levels and Intestinal Fat Absorption

Abstract

In an effort to identify novel high density lipoprotein (HDL) receptors expressed by the liver, a bacteriophage library expressing liver cDNA fragments was used to screen for clones that bind to HDL. The gene DENND5B showed significant enrichment for HDL binding over 3 cycles of biopanning, with negative selection against low density lipoprotein binding. Although little is known about the recently described DENN-domain containing family of proteins, some members act as guanine nucleotide exchange factors involved in the activation of Rab proteins and membrane trafficking. To examine the influence of DENND5B on HDL physiology, a DENND5B knockout mouse was generated using a custom zinc finger nuclease. DENND5B -/- mice had decreased plasma total cholesterol (-30.9%, p < 0.0001) and phosphatidylcholine (-31.4%, p < 0.0001). When plasma was analyzed by size-exclusion chromatography, the lipid effects were attributed entirely to decreased HDL. When fed western diet for 4 months, DENND5B -/- mice showed significantly smaller increases in plasma lipids and body weight compared to wild type mice. After sacrifice, DENND5B -/- mice were found to have a distended small intestine that was whitish in color. To evaluate fat absorption, mice were given an oral gavage of vegetable oil (10 uL/gram body weight) and plasma lipids were measured at 0, 2, and 4 hours post-gavage. DENND5B -/- mice had significantly reduced plasma triglyceride (p < 0.05) and free fatty acids (p < 0.01) in response to oil gavage compared to wild type mice. To identify a possible role for this gene in human lipoprotein metabolism, we examined gene expression data from 5,458 participants in the Framingham Heart Study. Consistent with the mouse data, a significant negative relationship exists between DENND5B expression and plasma HDL-C (p = 4.82 x 10 -5 ). In summary, using a phage display screening approach, we have identified a novel gene associated with circulating HDL levels in mice and humans. Additionally, in a homozygous knockout mouse model, a defect in intestinal fat absorption is present. Future studies will be aimed at evaluating the influence of this gene on HDL function, fat absorption, and atherosclerotic disease burden.