DENND5B Regulates Intestinal Triglyceride Absorption and Body Mass

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.

(GEF) activity toward Rab GTPases and the Rab specificity of DENN proteins varies among family members 9 . Based on this function, it has been proposed that DENN proteins are involved in the regulation of intracellular vesicular transport pathways. In mice, Dennd5b is expressed at relatively high levels in liver, small intestine, and brain 10 (Supplemental Fig. 1A).
In the current study, we generated a Dennd5b knockout mouse and found that the mice have decreased absorption of dietary TG due to a post-Golgi defect in chylomicron secretion. Additionally, we tested the hypothesis that DENND5B is important in human lipid metabolism using two independent exome sequencing cohorts.

Methods
Generation of Dennd5b −/− mice. A custom zinc finger nuclease (ZFN) was generated to target an exonic sequence within the first third of the Dennd5b gene. The ZFN RNA (20-40 ng/uL) was administered to fertilized B6CBAF1 mouse embryos by pronuclear injection and embryos were implanted into females. Resulting pups were genotyped, using PCR and Sanger sequencing. A Dennd5b −/− line with a biallelic 19 bp deletion was established. This mutation predicts a frameshift and early termination due to the introduction of a stop codon. Wildtype mice (WT) on the same background were used as controls for all experiments. Mice were fed standard chow (Envigo 7017) or western diet (WD) with 42% calories from fat (Harlan TD.88137). Western diet studies were initiated in mice beginning at 2 months of age. For all other experiments mice used were between 2-4 months of age and controls were age matched (within one week). All animal protocols and procedures conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the National Heart Lung and Blood Institute ACUC.

Blood collection and plasma lipid analyses.
For all experiments, mouse blood was collected by retro-orbital bleed, using heparinized capillary tubes and added to tubes containing EDTA. Plasma was obtained by centrifugation at 3,000 rpm for 20 min. at 4 °C. Plasma lipids were measured using colorimetric enzymatic assays (Wako Diagnostics). Size-exclusion chromatography separations of plasma lipoproteins were performed on an Akta Pure instrument equipped with two Superose 6 (GE Healthcare) columns arranged in series. Plasma (100 µL) was run over columns at a flow rate of 0.5 mL/min in Tris buffer (10 mM Tris, 150 mM NaCl, 0.5 mM EDTA, 0.01% sodium azide) and 0.5 mL fractions collected.
Vegetable oil (10 uL/g body weight) was administered by oral gavage, using a blunt ball-tipped syringe. Plasma was collected as described above, at time points indicated, and triglyceride measured by enzymatic assay. VLDL secretion assay. Mice were fasted overnight (16 hours) and baseline blood collected prior to retroorbital injection (6 µL/g body weight) of Tyloxapol in saline (5% wt:vol). Blood was collected from mice by retroorbital bleed from eye opposite to injection site at 1, 2, and 4 hours post-injection. Plasma was analyzed for triglyceride content by enzymatic assay.
Hepatic lipid analysis. Liver tissue harvested from PBS perfused mice was snap frozen with liquid nitrogen and stored at −80 °C until lipid extraction. A portion of liver (80-100 mg) from the lateral left lobe was used for lipid extraction. Tissue was added to 1 mL 0.9% NaCl, minced and lipids collected by chloroform methanol extraction. Chloroform layer containing lipid was dried under nitrogen gas. Dry lipid was solubilized in hexane isopropanol (3:2, v:v) and a small volume used for measurement of cholesterol, triglyceride, and free fatty acids by enzymatic assay. All measurements were adjusted to the starting liver tissue mass.
Fecal lipid analysis. Feces were collected from mice over a 3-day period and dried before measuring total mass. About 250 mg of dry feces was pulverized, using a mortar and pestle, and total lipids were isolated by chloroform methanol (2:1, v:v) extraction. Chloroform layer containing lipid was dried under nitrogen gas and weighed to determine total lipid mass. Dry lipid was solubilized in hexane isopropanol (3:2, v:v) and a small volume used for measurement of cholesterol, triglyceride, and free fatty acids by enzymatic assay (All from Wako Diagnostics). All measurements were adjusted to the starting fecal mass. electron microscopy of small intestine. Mice were sacrificed by cervical dislocation and a 1 mm section of duodenal small intestine tissue was quickly harvested beginning 1 mm distal to the pyloric sphincter of the stomach. Tissue was immediately placed in Karnovsky's fixative and chopped into cubes 1 mm 3 or less, post-fixed in osmium tetroxide, dehydrated and embedded in Epon for sectioning. Sections on uncoated grids were imaged on a JEOL JEM 1200EXII transmission electron microscope.
Aortic atherosclerosis lesion area quantification. Mouse aortas, from base of aortic arch to the femoral branch, were harvested and fixed in fresh 4% paraformaldehyde for 5 minutes before staining with Sudan IV (0.5%, wt:vol) for 15 minutes. After staining, aortas were destained for 15 minutes with 80% ethanol and stored in water. Scissors were used to open the aortas for en face analyses. Color images were taken along the full length of the aorta and imaging software was used to quantify stained lesions. Atherosclerotic lesion area was calculated as (stain positive area/total aortic surface area)*100.
DENND5B genotype association studies in Humans. The ClinSeq ® exome sequencing cohort 11 comprising 621 participants with reliable sequence data for the DENND5B Arginine 52 codon (variant rs4930979), were used for analysis. Body mass and blood lipid parameters were evaluated across homozygous reference, www.nature.com/scientificreports www.nature.com/scientificreports/ heterozygous, and homozygous variant genotypes for the c.155G > A; p.(R52K) variant by ANOVA with Tukey correction for multiple comparisons.
The Mayo Vascular Disease Biorepository (VDB) was used for replication analyses 12 . Genotyping of 9,274 VDB participants was performed, using genome-wide SNP arrays on Illumina platforms. Genotype data were imputed using Michigan Imputation Server 13 based on a human reference consortium (HRC, r1.1) panel 14 . Genotype association tests for lipid levels and BMI were based on linear regression analyses assuming an additive effect with adjustment for age, sex and the first two principal components.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Results
Dennd5b −/− mice have low plasma HDL. A Dennd5b −/− mouse line was generated, using a zinc finger nuclease. This approach resulted in a 19 bp deletion (Fig. 1A), predicting a frameshift mutation and early termination signal. Mice were fertile with outwardly normal morphology. Measurement of plasma lipids in Dennd5b −/− mice revealed a significant reduction in plasma total cholesterol (TC) and phospholipids (PL) but not triglyceride (TG) (Fig. 1B-D). Reduced lipids were only present in homozygous mice, not in heterozygous, and the effect was greater in females than in males (−30% vs. −20%). Because female Dennd5b −/− mice demonstrated a more prominent plasma lipid phenotype, female mice were used for subsequent experiments. Size-exclusion chromatography of mouse plasma demonstrated that the reduction in lipids was attributed entirely to a reduction of HDL-sized lipoproteins (Fig. 1E,F). Nuclear magnetic resonance analyses of lipoprotein particle concentrations indicated a 22% reduction in total HDL particle number (HDL-P), which was entirely due to a lower concentration of medium sized HDL particles (Fig. 1G). Large HDL particle numbers were not affected. Dennd5b is involved in post-Golgi chylomicron secretion by enterocytes. To gain insight into the mechanism of the involvement of Dennd5b in intestinal TG absorption, transmission electron microscopy was used to examine duodenal enterocytes in wildtype (WT) and Dennd5b −/− mice. Imaging of tissue at various time points after oral oil gavage allowed for visualization of the sequential steps of chylomicron secretion. At 1 hour post-gavage, fusion events between chylomicron secretory vesicles and the plasma membrane result in secretion of chylomicrons by WT enterocytes (Fig. 2H). In Dennd5b −/− mice, these fusion events are rare and very few extracellular chylomicrons are observed (Fig. 2I). At 2 hours, WT enterocytes have cleared the majority of lipid (Fig. 2J); however, there is significant accumulation of fat in Dennd5b −/− enterocytes in the form of vesicle-bound chylomicrons and some lipid droplets (Fig. 2K). A more detailed series of electron micrographs of enterocytes taken at various time points after oral oil gavage are presented in Supplemental Fig. 2. These images revealed similar lipid droplet formation, pre-chylomicron formation in the ER lumen, pre-chylomicron transport vesicles (PCTV's), chylomicron maturation in the Golgi and budding of chylomicron secretory vesicles (CSVs) from the Golgi in wildtype and Dennd5b −/− mice. However, in contrast to wildtype mice, Dennd5b −/− enterocytes exhibited massive accumulation of CSV's, which appeared to be unable to fuse with the basolateral plasma membrane. Whereas WT mice secreted chylomicrons into the intercellular space and the lamina propria by 1 hour after gavage, chylomicron secretion by Dennd5b −/− enterocytes was rarely observed. Lower magnification electron micrographs allowed for visualization of the extent of lipid accumulation in the enterocyte (Supplemental Fig. 3). Together, these findings support a role for Dennd5b in post-Golgi transport of chylomicron secretory vesicles. Enteric lipid accumulation was even observed in Dennd5b −/− mice that did not receive an oil gavage and had undergone an overnight period of fasting (Fig. 2L,M). In Dennd5b −/− enterocytes, electron dense structures similar to intracellular digestive vesicles were observed (Fig. 2M, red arrow and Supplementary Fig. 4), which may represent an alternative pathway for the removal of excess intracellular TG by autophagy.

Dennd5b −/− mice are resistant to western diet induced weight gain and changes in plasma lipids.
Dennd5b −/− mice and age-matched wildtype controls were placed on western diet (WD, 42% calories from fat and 0.2% cholesterol), beginning at 2 months of age. The rate of body weight gain in Dennd5b −/− mice was about half the rate of WT mice (Fig. 3A). After 4 months on WD, the body weight of Dennd5b −/− mice was 30% lower than WT mice. The difference in body weight was explained by a shift in body mass composition, with Dennd5b −/− mice having lower fat mass and greater lean mass (Fig. 3B). On standard chow diet, WT and knockout mice maintain similar body weight ( Supplementary Fig. 5). On WD, fecal lipids were measured and although total fecal mass was increased in Dennd5b −/− mice ( Fig. 3C and Supplementary Fig. 6A), significant increases in total lipid content were not detected, although there was a trend toward increased total lipids in Dennd5b −/− feces (Supplementary Fig. 6B). Measurement of specific lipid components revealed that fecal total cholesterol and triglyceride content were not different but fecal free fatty acid content was significantly increased in the www.nature.com/scientificreports www.nature.com/scientificreports/ knockout mice ( Supplementary Fig. 6C-E). In plasma, TC and PL increased compared to baseline values in WT mice and there was no change in triglyceride. However, Dennd5b −/− mice were resistant to WD-induced plasma lipid increases (Fig. 3D-F). While on WD, WT mice maintained a higher amount of HDL and accumulated a larger-sized population of cholesterol-rich lipoprotein, likely small LDL, that was absent in Dennd5b −/− mice (Fig. 3G,H). The favorable plasma lipid and body composition profile in Dennd5b −/− mice suggested a possible protective effect against diet-induced atherosclerosis. Analyses of atherosclerotic lesion area in mice after 4 months on WD showed significantly lower plaque burden in Dennd5b −/− mice compared to wildtype controls (Fig. 3I).
Dennd5b −/− mice compensate for lack of dietary lipid by increasing hepatic VLDL production. To evaluate the effect of Dennd5b knockout on hepatic lipid metabolism in mice on WD, we extracted lipid from liver tissue. TC and FFA were significantly reduced in Dennd5b −/− mouse liver and there was a trend www.nature.com/scientificreports www.nature.com/scientificreports/ toward decreased TG (Fig. 3J-L). The VLDL production by the liver was evaluated by measuring TG appearance in plasma of fasting mice after intravenous tyloxapol injection. Dennd5b −/− mice had a 21% increase in rate of VLDL associated TG accumulation compared to WT (slope = 7.5 vs 6.18, p = 0.0098) (Fig. 3M).  (Fig. 4A,C). The effect on BMI was due to increased body weight, no difference in height was observed. The p.(R52K) variant did not influence these measures in male participants (Fig. 4B,D) and did not correlate with plasma lipids for either gender in this cohort.
In an independent, larger cohort (Mayo Vascular Disease Biorepository, VDB), p.(R52K) (MAF = 0.3984) was associated with BMI (β = −0.179, P = 0.041, n = 8,303). In this dataset, another DENND5B variant p. (H487N) (rs1056320, MAF = 0.1072) was associated with LDL-C (β = 2.03, P = 0.0064, n = 8,571); however, this variant was www.nature.com/scientificreports www.nature.com/scientificreports/ 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. (2019) 9:3597 | https://doi.org/10.1038/s41598-019-40296-0 www.nature.com/scientificreports www.nature.com/scientificreports/ not significantly associated with BMI (β = −0.217, P = 0.11, n = 8,303). One explanation for the differing metabolic presentations associated with these variants is that they have distinct influences on protein function due to their location in the protein. Perhaps DENND5B performs more than one function with influence on metabolism.

Discussion
Overall, these data demonstrate a role for Dennd5b in murine enterocyte chylomicron secretion. In particular, Dennd5b appears to be critical in the Golgi to plasma membrane transport of chylomicron secretory vesicles. The metabolic consequence of genetic disruption of Dennd5b in mice is resistance to western diet induced increases in plasma lipids and body weight. Consistent with the observed phenotype in mice, we found that human variation in DENND5B is also associated with body weight and plasma lipids.
By electron microscopy, we were able to observe many of the key steps in the secretory pathway of chylomicrons. This allowed us to pinpoint the step of impairment in Dennd5b −/− mice at the level of Golgi to plasma membrane transport of chylomicron secretory vesicles. The mechanisms of pre-Golgi and intra-Golgi processing of chylomicrons are well understood 6 ; however, post-Golgi steps in the chylomicron secretory pathways are relatively uncharacterized. The Dennd5b −/− mouse generated in this study, therefore, provides a unique and novel tool for further exploration of this important metabolic function.
Increased free fatty acid content in the feces of Dennd5b −/− mice on WD supports the conclusion that impaired chylomicron secretion by the enterocyte results in an overall decreased absorption of dietary triglycerides. The appearance of electron dense, layered lipid structures by electron microscopy in Dennd5b −/− mice after oil gavage suggests that the enterocyte does initially absorb fatty acid, but it accumulates intracellularly and shunts it to an autophagolytic pathway. This alternative pathway for triglyceride that is not secreted by enterocytes into the lymph may explain the lack of weight gain in the knockout mice on WD. In addition, unabsorbed free fatty acids in the intestine may likely be metabolized by gut bacteria, which may then have consequences on the gut microbiome and overall metabolism. Because chylomicrons are also important mediators of cholesterol absorption, the lack of increased fecal cholesterol excretion in Dennd5b −/− mice was unexpected. Further studies will be required to elucidate the specific pathways involved in the metabolic disturbances observed in this model. www.nature.com/scientificreports www.nature.com/scientificreports/ Increased hepatic VLDL production by Dennd5b −/− mice may be a compensatory mechanism for peripheral triglyceride distribution under the circumstance of reduced availability of dietary lipids. This observation also prompts the conclusion that Dennd5b is not essential for the secretion of all apoB containing lipoproteins. This may suggest the existence of alternative post-Golgi mechanisms for hepatic VLDL secretion. Additionally, reduced lipid accumulation in Dennd5b −/− liver could support a potential protective effect against non-alcoholic fatty liver disease.
Other members of the DENN domain-containing family of proteins may shed light on the details of DENND5B's mechanism of action in chylomicron secretion. The DENN domains commonly contain GEF activity for Rab proteins, which are important effectors of intracellular vesicular trafficking 15 . Interestingly, one report that has characterized the GEF activities of the DENN has family of proteins found DENND5B to have GEF activity for Rab39 9 . Rab39 is also not well documented in the literature but has been reported as a Golgi-associated protein with involvement in intracellular trafficking 16 . As is true of all secretory processes, it is likely that there are many potential mediators involved in this pathway of Golgi to plasma membrane transport and fusion of chylomicron secretory vesicles. Future studies on the GEF activity of DENND5B may reveal important insight into the regulation of chylomicron secretory processes.
It is noteworthy that the reported mouse phenotype was only detected in homozygous knockouts. This suggests that Dennd5b-related dyslipidemia is inherited in an autosomal recessive pattern. In ClinSeq ® , only subjects homozygous for the p.(R52K) minor allele had reduced BMI and abdominal circumference. Several in silico tools predict that this variant is benign (Mutation taster 0.0002 probability of pathogenicity, Polyphen2 0.0002 probability of pathogenicity, SIFT score 0.267 (tolerated)). That we observed a significant influence of this variant on relevant traits suggests either that the p.(R52K) variant itself has deleterious effects on DENND5B function in spite of the in silico predictors or that this variant is in linkage disequilibrium with another pathogenic variant in DENND5B.
In our mice, we confirmed GeneAtlas expression data indicating that Dennd5b is highly expressed in liver and intestine (Supplemental Fig. 1A). In the small intestine, expression levels were highest in duodenum and decreased in distal small intestine (Supplemental Fig. 1B). We observed a gender difference in hepatic Dennd5b expression. Female mice have significantly higher expression of Dennd5b in the liver, this may be related to the greater impact of Dennd5b deletion on circulating plasma lipid levels in female mice. Gender differences also appear to modulate the phenotype of this gene in humans. The effect of the p.(R52K) variant was only observed in females. Interestingly, the ExAC database 17 shows only two heterozygous loss of function variants amongst ~60,000 individuals, which yields a pLI (Probability of Loss of Function Intolerance) score of 1.00 (the highest possible score). These data suggest that homozygous loss of function variants in the human cause a highly deleterious phenotype, which we hypothesize to be an essential function related to intracellular transport.
By influencing plasma lipids and body weight, DENND5B might be expected to affect cardiovascular disease risk, specifically atherosclerosis. In the knockout mouse, there was a reduction of total atherosclerosis burden. Although we did observe decrease atherosclerosis in the Dennd5b −/− mouse compared to WT mice, the extent of atherosclerosis in WT mice was quite small. Future studies on Dennd5b −/− crossed with ApoE −/− or Ldlr −/− are needed to better examine the role of Dennd5b in atherogenesis. There is also evidence of a possible association of DENND5B variants and atherosclerosis in humans. Levy et al. have found that miR-150-5p, which has a potential causal association with coronary heart disease, may act by regulating expression of DENND5B, among other target genes 18 . Furthermore, in vitro studies using the Huh7 human liver cell line have shown that another miRNA, miR-223, known to be associated with inflammation and cholesterol metabolism, causes significant downregulation of DENND5B 19 . Another study has reported an association between expression of DENND5B in whole blood and coronary artery disease in humans 20 . These studies suggest a complex regulatory network for DENND5B expression in humans and further highlight the potential importance of this gene in cardiovascular disease.
In summary, DENND5B was identified to play an important role in dietary lipid absorption (i.e., Golgi to plasma membrane trafficking of chylomicron secretory vesicles) and was found to also effect body weight, plasma lipid metabolism, and atherosclerotic cardiovascular disease in knockout mice. Further investigation into the mechanistic details of this activity may provide additional targets and alternative approaches to the treatment of metabolic and cardiovascular disease in humans.