Abstract Body: Background: Elevated plasma triacylglycerides (TAGs) are a risk factor for atherosclerosis and are regulated by dietary absorption via the small intestine and endogenous production by the liver. Our laboratory reported that Dennd5b-deficient mice are protected from diet-induced hepatic steatosis, hyperlipidemia, and atherosclerosis. Electron microscopy imaging of intestinal epithelium of Dennd5b^-/- mice revealed significant lipid accumulation in intracellular chylomicron secretory vesicles, suggesting a post-Golgi chylomicron secretion defect. Since DENND5B is expressed by both triglyceride-rich lipoprotein (TRL)-secreting organs (liver and small intestine) in Humans, we hypothesized that DENND5B disruption attenuates TRL secretion in both of these tissues.

Methods and Results: To determine if DENND5B disruption impairs TRL secretion in human cells, we generated DENND5B^-/- intestinal epithelial (Caco-2) and hepatocyte (HepG2) cell lines using CRISPR. Western blots verified absence of DENND5B in DENND5B^-/- cells. Cellular and secreted TAG was measured from DENND5B^+/+ and DENND5B^-/- cells in growth media and after oleic acid loading. DENND5B disruption significantly reduced TAG secretion in both Caco-2 and HepG2 cells compared to DENND5B^+/+ controls (Caco-2: -67.8%, p<0.0001; HepG2: -91.0%, p<0.0001) without affecting cellular TAG content. RNA-seq analysis revealed altered lipid and lipoprotein metabolism gene expression in DENND5B^-/- cells. Immunofluorescent microscopy of DENND5B^-/- HepG2 cells showed striking APOB accumulation consistent with impaired VLDL secretion. To gain insight into the functional role of DENND5B in TRL secretion, we transfected HepG2 cells with mCherry and GFP-tagged DENND5B plasmids. Confocal microscopy showed DENND5B localization to cytoplasmic vesicles and associated with cytoskeletal structures. Consistent with this, co-immunoprecipitation of DENND5B from HepG2 cells showed enrichment of proteins involved in motor protein binding and microtubule-membrane tethering.

Conclusions: These data support the hypothesis that DENND5B acts in TRL secretion from human enterocytes and hepatocytes. Our findings suggest a mechanism where DENND5B facilitates vesicular transport of TRL’s along the cytoskeleton. Understanding the mechanistic details of this process may provide novel targets for regulation of plasma TAG.

This research was funded by the American Heart Association Pre-Doctoral Fellowship to Olivia Hage, and NIH grant R01DK133184.