Abstract 2044: Disruption Of DENND5B Attenuates Triglyceride-rich Lipoprotein Secretion From Human Enterocytes And Hepatocytes

Abstract

Background and Hypothesis: Elevated plasma triacylglycerides (TAGs) are an established risk factor for atherosclerotic cardiovascular disease, and are regulated by dietary absorption via the small intestine and endogenous production by the liver. Our laboratory formerly reported that Dennd5b -deficient mice are protected from diet-induced hepatic steatosis, hyperlipidemia, and atherosclerosis. Electron microscopy imaging of the intestinal epithelium of Dennd5b -/- mice reveals significant lipid accumulation in intracellular chylomicron secretory vesicles, suggesting a post-Golgi chylomicron secretion defect. Given that Dennd5b is expressed by the two major triglyceride-rich lipoprotein (TRL)-secreting organs (liver and small intestine), we hypothesized that disruption of DENND5B attenuates TRL secretion in both of these cell types in humans. 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 gene editing. Western blots verified DENND5B protein reduction in DENND5B -/- cells. The impact of DENND5B deficiency on TRL secretion was quantified by measuring the cellular and secreted TAG content of DENND5B +/+ and DENND5B -/- cells under standard growth conditions and after oleic acid loading. Caco-2 cells were differentiated on membrane supports for 21 days prior to experiments. DENND5B -/- Caco-2 cells secreted significantly less TAGs compared to wild-type Caco-2 cells (-67.8%, p<0.0001). DENND5B -/- HepG2 cells also had significantly reduced TAG secretion (-91.0%, p<0.0001). Disruption of DENND5B in both cell lines reduced TAG secretion without affecting cellular TAG content. Immunofluorescence microscopy showed intracellular DENND5B protein localization consistent with secretory pathway involvement with detection on a perinuclear structure (likely Golgi), cytosolic vesicles, and plasma membrane. Conclusions: These studies suggest that DENND5B plays a role in TRL secretion from human enterocytes and hepatocytes and provide new model systems for investigating the molecular function of DENND5B in TRL secretion from human cells.