DENND5B Regulates Intestinal Triglyceride Absorption and Body Mass

Abstract

Regulation of lipid absorption by enterocytes can influence metabolic status in humans and contribute to obesity and related complications. The intracellular steps of chylomicron biogenesis and transport from the Endoplasmic Reticulum (ER) to the Golgi complex have been described, but the mechanisms for post-Golgi transport and secretion of chylomicrons have not been identified. Using a newly generated Dennd5b^-/- mouse, we demonstrate an essential role for this gene in Golgi to plasma membrane transport of chylomicron secretory vesicles. In mice, loss of Dennd5b results in resistance to western diet induced obesity, changes in plasma lipids, and reduced aortic atherosclerosis. In humans, two independent exome sequencing studies reveal that a common DENND5B variant, p.(R52K), is correlated with body mass index. These studies establish an important role for DENND5B in post-Golgi chylomicron secretion and a subsequent influence on body composition and peripheral lipoprotein metabolism.

Figure 1

Knockout of Dennd5b in mice results in reduced plasma high density lipoprotein lipids and particle number. (A) Genetic knockout of Dennd5b in mice using zinc-finger nuclease resulted in a 19 bp deletion. Genotyping strategy utilizes PCR amplification of a DNA fragment containing the cut site and exploits loss of Bpil restriction site in mutant mice. (B–D) Total cholesterol, phospholipid, and triglyceride in plasma of wildtype (green), heterozygous (blue), and homozygous (red) mice by gender. n = 6–12/group on chow diet. (E,F) Size-exclusion chromatography analyses of plasma lipoproteins in wildtype, heterozyous, and homozygous female mice. Each trace represents a pooled sample from n = 3/group on chow diet. (G) High density lipoprotein particle number was measured by nuclear magnetic resonance on a Vantera Lipoprotein Analyzer. n = 3 pools of 5 female mice/group on chow diet. Statistical analyses were performed using 2-way ANOVA with Tukey post hoc tests (**p < 0.01, ***p < 0.001, ****p < 0.0001). All values are mean ± standard deviation.

Figure 2

Dennd5b^−/− mice have enlarged small intestine and reduced triglyceride absorption due to impaired chylomicron secretion by enterocytes. (A) Picture of duodenal segments from overnight fasted wildtype and Dennd5b^−/− mice. (B) A 25 mm segment of duodenal small intestine, beginning 20 mm distal to pyloric sphincter, was cut longitudinally and laid flat for imaging and surface area calculation. Lumenal surface area was calculated as length × width of the segment in image pixels. n = 6/group. ***p < 0.0001 by unpaired t-test. (C) Mice were given an oral gavage of vegetable oil (10 uL/g of body weight) and appearance of triglyceride in the plasma was measured at baseline, 2, and 4 hours post-gavage. n = 10/group. *p = 0.01 for wildtype vs knockout using 2-way ANOVA with Sidak post hoc test. (D,E) Masson stained duodenal sections from wildtype and Dennd5b^−/− mice 2 hours after oral oil-gavage. (F,G) Immunofluorescence stained duodenal sections 2 hours after oral oil-gavage. DAPI nuclear stain (Blue) and anti-apoB (Red). (H,I) Electron micrographs of duodenal tissue at one hour after oral oil-gavage. (J,K) Electron micrographs of duodenal tissue at two hours after oral oil-gavage. (L,M) Electron micrographs of duodenal tissue after an overnight fasting period. All values are mean ± standard deviation. All mice used in these experiments were female.

Figure 3

Knockout of Dennd5b confers resistance to western diet induced weight gain, plasma and liver lipid increases, and atherosclerotic lesion development. (A) Body weights of wildtype and Dennd5b^−/− mice during 4-months on western diet. n = 6/group. *p < 0.0001 by 2-way ANOVA with Sidak post hoc test. (B) NMR body composition analyses of wildtype and Dennd5b^−/− mice after 4-months on western diet. n = 6/group. **p < 0.01 by 2-way ANOVA with Sidak post hoc test. (C) Feces were collected from mice over a 3-day period and dried before measuring total mass. n = 6–8/group. **p < 0.01 by unpaired t-test. (D–F) Plasma total cholesterol, phospholipid, and triglyceride were measured during 4-months on western diet. *p < 0.01 t-test with multiple comparisons correction by the Holm-Sidak method. (G–H) Size-exclusion chromatography analyses of plasma lipoproteins in wildtype and homozygous mice after 4-months on western diet. Each trace represents a pooled sample from n = 3/group. (I) Quantification of lipid rich atherosclerotic lesions in the aortas of mice after 4-months on western diet. Aortas were harvested, stained with Sudan IV, and mounted for en face analyses. Plaque area was calculated as % of total aorta area cover by positive Sudan IV staining. *p < 0.05 by unpaired t-test. (J–L) Lipids were extracted from liver after 4-months on western diet and total cholesterol, triglyceride, and free fatty acids were measured. n = 6/group. *p < 0.05 by unpaired t-test. (M) VLDL production was measured by giving mice a retroorbital injection of tyloxapol and monitoring the appearance of triglyceride in the plasma over time. The rate of VLDL production was calculated from the slopes of the lines. All values are mean ± standard deviation. All mice used in these experiments were female.

Figure 4

A common DENND5B genetic variant is associated with body mass index and abdominal circumference in humans. (A,B) An evaluation of body mass index (BMI) among wildtype, heterozygous, and homozygous carriers of the p.(R52K) rs4930979 variant in females (A) and males (B). (C,D) An evaluation of abdominal circumference among wildtype, heterozygous, and homozygous variant genotypes for the p.(R52K) variant in females (C) and males (D). Numbers at the base of each bar are the group mean. Statistical comparisons were performed by one-way ANOVA with post hoc correction by the Tukey method. *p < 0.05, **p < 0.01, n.s. = not significantly different. All values are mean ± standard deviation.