Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency

Abstract

Background & aims: Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids.

Methods: We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7.

Results: In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids.

Conclusions: We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.

Keywords: 3-D Culture Model; CDD; Genomic; PLE.

Graphical abstract

Figure 1

Pedigrees, mutations, and genetic location of 6 families with DGAT1 deficiency. (A) Pedigrees of families with DGAT1 deficiency and chromatograms showing mutation in affected patients. Filled shapes indicate affected individuals, half-filled are heterozygous for mutation indicated, and empty shapes indicate WT. (B) Exonic scheme of DGAT1 showing mutations identified in this study in black and previously identified mutations in red., , ,

Figure 2

DGAT1 protein expression in patient-derived material. (A) Immunohistochemistry of DGAT1 in control and patient 1 and patient 2 ileal and duodenal biopsy, respectively. (B) Western blot showing lack of DGAT1 and DGAT2 in patient 3 fibroblast lysate, but normal expression of DGAT1 in healthy control fibroblasts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Western blot showing lower expression of potentially nonfunctional DGAT1 protein and normal DGAT2 protein level from Epstein-Barr virus–derived B lymphoblastoid cell line of patient 4, a parent, and a healthy control. GAPDH was used as a loading control. (D) Western blot for DGAT1 protein expression in undifferentiated (EM) and differentiated (DM) organoids from 2 healthy controls and patients 7 and 8. HSP90 was used as loading control. Results are representative of 3 independent experiments.

Figure 3

Loss of DGAT1 results in decreased lipid droplet formation in organoids. (A) Immunofluorescent images of 4′,6-diamidino-2-phenylindole (DAPI) (blue) and LD540 (yellow) staining of organoids from healthy control, DGAT1 mutant patient 8 (P8) and DGAT1^KO organoids after 17-hour incubation with vehicle control (BSA), 1 μM OA, or 1 μM OA + 0.1 μM DGAT1 inhibitor (OA+DGAT1i). Representative images of 3 healthy controls, 3 patients (patients 7–9), and 3 CRISPR/Cas9 genome-edited DGAT1-knock-out (DGAT1^KO) organoids. (B) Representative histograms of SSC and LD540 staining in organoids from controls, patients, and DGAT1^KO organoids as described in (A). Upon OA stimulation, control organoids accumulate lipid droplets and show increased SSC and LD540, which was severely reduced in patient-derived and DGAT1^KO cells. Mean fluorescence intensity (MFI) of SSC and LD540 was plotted for n = 3 per group. Statistical analysis was done using a 2-way analysis of variance with Tukey’s multiple comparison test. Mean ± SD is indicated; * P ≤.05, *** P ≤.001.

Figure 4

Loss of DGAT1 results in decreased lipid droplet formation in fibroblasts. (A) Left: Representative contour plots for forward (FSC-A) and SSC-A of patient 3 and 2 control fibroblasts with and without OA addition. Right: Mean SSC-A of 5 technical replicates. (B) Left: Representative histogram of Nile Red mean fluorescence intensity (MFI) of patients 3 and 2 control fibroblasts with and without OA addition. Right: MFI of 5 technical replicates. (C) Western blot showing retroviral-mediated delivery of exogenous DGAT1 and DGAT2 on patient 3 fibroblasts. (D) Mean SSC-A and (E) MFI of Nile Red staining on patient 3 fibroblasts reconstituted with empty vector (EV), WT DGAT1, or WT DGAT2. Statistical analysis was done using 2-way analysis of variance with Tukey’s multiple comparison test. Mean ± SD is indicated; **P< .01 or ****P < .0001.

Figure 5

Loss of DGAT1 results in decreased triglyceride (TG) formation. (A) Organoids from 3 healthy controls and patients 7, 8, and 9 were grown in EM for 7 days. Lipid extracts were isolated and run by thin layer chromatography to determine levels of diacylglycerol (DG) and triglyceride (TG). Relative positions were indicated by reference samples for DAG and TG. (B) Ratio of intensity of TG over DAG for each sample. Statistical analysis was done using a Mann-Whitney U test. Mean ± SD is indicated, ∗P < .05.

Figure 6

Loss of DGAT1 results in increased sensitivity to lipotoxic stress. Organoids of 3 healthy controls and 3 patients (7–9) were grown in EM and incubated overnight with a range of oleic acid (OA) concentrations. (A) Representative images showing brightfield and propidium iodide staining for cell death of the organoids after incubation with OA. (B) Caspase-Glo 3/7 assay for apoptotic cells after incubating organoids with OA. Samples were normalized for vehicle control values and the maximum value for each sample was set to 100% assay response. Median lethal dose (LD₅₀) was calculated by regression analysis. Statistical analysis on LD₅₀ values was done using a Student’s t test, ***P ≤ .001.

Supplementary Figure 1

Histologic and endoscopic characteristics of intestines of DGAT1-deficient patients. (A) Colonoscopy (left) and electron microscopy (EM, right) revealed normal intestinal structures in patient 1. (B) Periodic acid-Schiff and EM of patient 7 were obtained during fat-free diet and do not show abnormalities. (C) Patient 10 shows cytosolic CD10 staining and lateral microvilli on EM.

Supplementary Figure 2

Hematoxylin-eosin (H&E) and immunohistochemical staining for DGAT1 in (A) healthy controls, (B) patient 2, and (C) patient 1.

Supplementary Figure 3

Family pedigrees and Sanger sequencing histograms of DGAT1 deficiency. Filled shapes indicate affected individuals, half-filled are heterozygous for mutation indicated, and empty shapes indicate wild type.

Supplementary Figure 4

Defective mRNA splicing in patient 3. (A) Gel showing aberrant DGAT1 transcripts in mRNA isolated from patient 3 fibroblasts with and without a 4-hour puromycin treatment. * denotes aberrant transcript. ACTB was used as control.

Supplementary Figure 5

DGAT1 c.629_631delCCT results in increased proteasomal degradation of DGAT1. Caco-2 cells were stably transfected with indicated constructs. (A) qRT-PCR analysis of DGAT1 mRNA expression, relative to HPRT1, in Caco-2 cells stably transfected with indicated constructs. Average and standard deviation of 3 independent experiments are shown. (B) Transfected Caco-2 cells were untreated or treated with 2 μM MG132 for 16 hours. Protein expression was determined by Western blot analysis using anti-Flag and anti-HSP90 antibodies. Results are representative of 3 independent experiments. (C) Caco-2 cells were transiently transfected with His-ubiquitin and indicated constructs and treated with 2 μM MG132 for 16 hours. Ubiquitination of DGAT1 was determined by a His pulldown and subsequent Western blot analysis using anti-Flag antibodies. Results are representative of 3 independent experiments.

Supplementary Figure 6

Generation of DGAT1 knockouts in healthy human intestinal organoids. (A) Targeting strategy for the generation of DGAT1 knockout organoids using CRISPR/Cas9 genome editing. (B) Schematic view of insertion-deletion mutations of 3 DGAT1^KO clones. Gray areas represent deleted sequences from the WT DGAT1 gene. (C) Western blot analysis of DGAT1 expression in DGAT1^KO organoids compared with healthy control 1 (HC1) and patient 8 (P8). Actin, loading control.

Supplementary Figure 7

Lipid droplet staining in Epstein-Barr virus–derived B-lymphoblastic cell line (EBV B-LCL) of patient 4 with and without OA. (A) Mean SSC-A and (B) Nile Red mean fluorescence intensity (MFI) staining of lipid droplet stained EBV B-LCLs.